Identification of Compounds Modifying a Cellular Response

ABSTRACT

The present invention relates to methods for identifying compounds capable of modulating a cellular response. The methods involve attaching living cells to solid supports comprising a library of test compounds. Test compounds modulating a cellular response, for example via a cell surface molecule may be identified by selecting solid supports comprising cells, wherein the cellular response of interest has been modulated. The cellular response may for example be changes in signal transduction pathways modulated by a cell surface molecule.

All patent and non-patent references cited in the application are herebyincorporated by reference in their entirety.

FIELD OF INVENTION

The present invention relates to a method and tools for extractinginformation relating to an influence, for example on a surface receptor,in particular an influence caused by contacting a receptor with asubstance linked to a solid support to which a cell expressing thesurface receptor is attached. In particular the method related to asolid support that allow chemical synthesis of individual substances onbeads of the solid support

The method of the invention may be used as a very efficient procedurefor testing or discovering the influence of a library of substances on aphysiological process, for example in connection with screening for newdrugs, testing of substances for toxicity, identifying drug targets forknown or novel drugs. Other valuable uses of the method and technologyof the invention will be apparent to the skilled person on the basis ofthe following disclosure

BACKGROUND OF INVENTION

Combinatorial synthesis of peptide as well as small-molecule librarieshas proven very useful as a method for generating vast numbers of highlydiverse compounds (see for example Comprehensive Survey of CombinatorialLibrary Synthesis: 2002 Roland E. Dolle J. Comb. Chem., 2003, pp693-753). To fully exploit this high capacity of combinatorial chemistryto produce huge numbers of compounds several technologies have beendeveloped that allow screening directly on the solid support (M. Meldal,1994, METHODS: A companion to methods of enzymology 6:417-424). In thefield of drug discovery such methods have successfully been applied forexample for the identification of enzyme modulators. The library can besynthesized on resin beads that each carry one specific compound, andthese “one-bead-one compound” libraries are then screened against thepurified biological component of interest (e.g. cellular proteins orpeptides),

Before progressing active compounds, identified though such procedure,further in the drug discovery process, the compound will have to bere-synthesized and tested for efficacy in a cell-based or in-vivo testsystem.

Novel ways to screen combinatorial libraries in a physiological morecorrect way are assumed to greatly accelerate the drug discoveryprocess, and show importance in areas like chemo-genomics andchemo-proteomics.

Screening of combinatorial libraries in intact cells have been done bycapturing mammalian or yeast cells together with a limited number ofresin-beads in a “nanodroplet” (Borchart et al. Chem Biol 1997 4:961).Compounds immobilized on the resin are released through disruption of aphoto-cleavable linker and the compound-associated effects on the intactcells are monitored.

In an alternative method the compounds are released through acidolysisresin-beads carrying the library members area are spread out on a lawnof mammalian cells, and the spatial localization of a cellular responseis monitored and beads in that region is isolated, and the remainingcompound is structure elucidated Jayawickreme et al, 1998, Combinatorialpeptide Library Protocols, Ed. Shmuel Cabilly, Humana Press, p.107-128).

WO03/038431 describes methods for screening combinatorial bead librariesby capturing cells from body fluids. Beads comprising a compoundenabling cells to adhere to said bead may be selected.

US2003/0059764 describes multiplexed cell analysis systems usingnon-positional or positional arrays of coded carriers.

SUMMARY OF INVENTION

It is of great importance to provide new and efficient methods foridentification of compounds influencing specific cellular processes. Inparticular, such methods wherein a very large quantity of candidatecompounds may be tested for a specific effect on a cell within arelatively short period of time.

It is therefore an object of the present invention to provide veryefficient procedures for testing or discovering the influence ofcompounds of a library on a physiological process in a cell. Inparticular, the methods provides means for testing very large numbers ofdifferent compounds for one or more physiological effects within arather short time period. This may be obtained by attaching living cellsto resin beads coupled to a test compound. The test compounds may thusinfluence physiological processes in said cells. Said influence(s) maybe detected and beads containing cells displaying the desiredinfluence(s) may be selected. Once selected the compounds coupled to theselected beads may be identified. These methods may for example be veryuseful in connection with screening for new drugs, testing of substancesfor toxicity, identifying drug targets for known or novel drugs.

Accordingly, it is a first objective of the invention to provide methodsof identifying a compound modifying at least one cellular response,wherein each cellular response is linked to different reporter systemsgenerating detectable outputs, said method comprising the steps of:

-   -   (a) Providing multiple resin beads capable of supporting growth        of cells, wherein each resin bead comprises one member of a        library of test compounds and wherein at least two beads        comprise different library members; and    -   (b) Attaching cells comprising said reporter system(s) onto said        resin beads; and    -   (c) Screening said resin beads for beads comprising cells        meeting at least one predetermined selection criterion, wherein        said selection criterion is linked directly or indirectly to a        detectable output; and    -   (d) Selecting beads comprising cells meeting said at least one        selection criterion; and    -   (e) Identifying said the library member, thereby identifying a        compound modifying said at least one cellular response.

DESCRIPTION OF DRAWINGS

FIG. 1A illustrates a method of identifying a resin bead comprising acompound influencing a cellular response linked to a reporter systemgenerating a fluorescent output. The method involves cultivating cellson resin beads, fixing cells, FABS calibration using a positive and anegative control, identification and isolation of positive hits.

FIG. 1B illustrates a method of identifying a resin bead comprising acompound influencing a cellular response linked to a reporter systemgenerating a fluorescent output detectable using a plate reader or imageacquisition analysis. The method involves 1) Grow cells on beads for 24hrs and Fix cells in EtOH, 2) Add app. 20 beads to each well andidentify hit wells using plate reader or image acquisition/analysis and3) Transfer beads from hit wells to a new 384 well plate—one bead/welland identify hit wells using plate reader or image acquisition. If forexample 500,000 beads are screened with 20 beads/well, approx, 25.000wells, i.e. approx. 68 plates must be screened. With a 0.1% hit rate,there will be approx. 500 hit wells comprising approx. 10,000 beads,which amounts to analysis of approx. 27 plates in the second round.Alternatively, positive beads may be picked directly (preferably withoutfixation) after the first identification using image acquisitionanalysis. The method may for example be used for analysis of expressionof a Cre-YFP construct.

FIG. 2A illustrates a multiplexed screen involving FABS and microscopy.The screen involves I) identification of positive hits by FABS asdisplayed in FIG. 1, followed by II) a step of microscopy identifyingresin beads comprising cells with an internal fluorescent signal. Thescreen could for example be a screen for Cre-YFP and MC4R-GFP or HA-MC4Rinternalisation, wherein I) Cre-YFP reporter hits are identified andisolated by FABS and II) MC4R-GFP or HA-GFP internalisation positivehits are picked.

FIG. 2B illustrates a multiplexed screen involving two FABS analysis.The screen involves I) identification of positive hits by FABS asdisplayed in FIG. 1, followed by II) a second FABS analysis. The screencould for example be a screen for Cre-YFP and HA-MC4R internalisation,wherein I) Cre-YFP reporter hits are identified and isolated by FABSinto a 10 ml. tube (see FIG. 1) and II) HA-MC4R internalisation hits areisolated (=low fluorescence).

FIG. 3 illustrates a plasmid map of pCRE-d2EGFP

FIG. 4A illustrates synthesis of Ac-His-(D)phe-Arg-Trp-NH₂.

FIG. 4B illustrates synthesis of Ac-His-(D)phe-Arg-Trp-Gly-PEGA₁₉₀₀

FIG. 4C illustrates synthesis of Fmoc-Dap(N₃)

FIG. 5 illustrates synthesis of the cyclic peptide of example 3

FIG. 6 a illustrates synthesis of a combinatorial library (6a) via anintramolecular N-acyliminium Pictet-Spengler reaction

FIG. 6 b illustrates synthesis of a combinatorial library (6b) via anintramolecular N-acyliminium Pictet-Spengler reaction

FIG. 7 illustrates spectra and structure determination by accurate massdifferences from single beads

FIG. 8 illustrates structure determination by accurate mass differencesfrom single beads

FIG. 9 illustrates a fragmentation pathway

FIG. 10 illustrates examples of an adhesion peptide displaying beadcovered with cells (U2OS).

FIG. 11 illustrates quantification of MC4R-GFP internalization on beads

FIG. 12 illustrates intracellular Ca²⁺ mobilization as visualised usingthe Flou4 probe.

FIG. 13 illustrates the aMSH induced CRE-YFP transcription in HEK293 andU2OS cells, respectively, expressing MC4.

FIG. 14 illustrates signal obtained from a subfraction of identifiedhits after functional screening (CRE-YFP) of a library.

FIG. 15 a is a picture of a bead with cells screened as described inExample 14a comprising the compound designated TEN-636-36-36.

FIG. 15 b illustrates QTOF MSMS analysis of the compound designatedTEN-636-33-26.

FIG. 16 illustrates MSMS analysis of material cleaved from a single beadof a library prepared as described in Examples 6a or 6b. Structureelucidation is given by [M+H]⁺, [M-Gly-AA1]⁺, and [M-Gly-AA₁AA₂]⁺.

DEFINITIONS

Naturally occurring amino acids are named herein using either their1-letter or 3-letter code according to the recommendations from IUPAC,see for example http://www.chem.qmw.ac.uk/iupac. If nothing else isspecified amino acids may be of D or L-form. In the description (but notin the sequence listing) 3-letter codes starting with a capital letterindicate amino acids of L-form, whereas 3-letter codes in small lettersindicate amino acids of D-form.

The term “a” as used herein, can mean one or more, depending on thecontext in which it is used.

In the present context, the term “green fluorescent protein” or (GFP) isintended to indicate a protein which, when expressed by a cell, emitsfluorescence upon exposure to light of the correct excitation wavelength(cf. [(Chalfie et al. 1994)]). “GFP” as used herein means any protein orfragment thereof capable of fluorescing when excited with appropriateradiation. This includes fluorescent proteins that are either naturallyoccurring or engineered and proteins that have been modified to befluorescent. Naturally occurring fluorescent proteins have been isolatedfrom the jellyfish, Aequorea vistoria, the sea pansy, Renillareniformis, Phialidium gregarium and Discosoma coral (W. W. Ward et al.(1982) Photochem. Photobiol, 35:803-808; Levine et al. (1982) Biochem.Physiol., 72B:77-85; Fradkov et al. (2000), FEBS Lett. 479:127-130).GFPs have also been engineered to emit different colors and to fluorescemore intensely in mammalian organisms (U.S. Pat. No. 5,625,048; WO97/28261; WO 96/23810; EP0851874; U.S. Pat. No. 6,172,188; WO01/98338).

A variety of Aequorea-related fluorescent proteins have been engineeredto have different excitation and emission spectra by modifying thenaturally occurring amino acid sequence (D. C. Prasher et al. (1992)Gene 111:229-233; Heim et al. (1994) Proc. Natl. Acad. Sci. USA 91:12501-12504; U.S. Pat. No. 5,625,048; WO 96/23810 and PCT/US97/14593).

The term “living cell” is used to indicate a cell which is consideredliving according to standard criteria for that particular type of cellsuch as maintenance of normal membrane potential, cell membraneintegrity and energy metabolism

The terms “image processing” and “image analysis” are used to describe alarge family of digital data analysis techniques or combination of suchtechniques which reduce ordered arrays of numbers (images) toquantitative information describing those ordered arrays of numbers.When said ordered arrays of numbers represent measured values from aphysical process, the quantitative information derived is therefore ameasure of the physical process.

The term “fluorescent probe” is used to indicate a fluorescent fusionpolypeptide comprising a GFP or any functional part thereof which is N-or C-terminally fused to a biologically active polypeptide as definedherein, optionally via a peptide linker consisting of one or more aminoacid residues, where the size of the linker peptide in itself is notcritical as long as the desired functionality of the fluorescent probeis maintained. A fluorescent probe according to the invention isexpressed in a cell and basically mimics the physiological behaviour ofthe biologically active polypeptide moiety of the fusion polypeptide.

The term “determining the fluorescence” is used to describe the processused to monitor a change in fluorescence properties.

The term “bioluminescence” is used to describe a process where light isproduced through a chemical reaction that natively is occurring in abiological system. For the reaction to occur at least two chemicals arerequired: the one that produces the light (called “luciferin”) and theother (called “luciferase”) that catalyzes the reaction. Sometimes theluciferin and luciferase are brought together in one single unit (called“photoprotein” an example of the last group is aequorin.

The term “FRET” is used to describe the occurrence of Fluorescenceresonance energy transfer between a fluorophore donor and an acceptorchromophore. It is a distance-dependent interaction between theelectronic excited states of two fluorophores in which excitation istransferred from a donor fluorophore to an acceptor chromophore withoutemission of a photon. The efficiency of FRET is dependent on the inversesixth power of the intermolecular separation, making it useful overdistances comparable with the dimensions of biological macromolecules.Thus, FRET is an important technique for investigating interactionsbetween cellular molecules for example complex formation.

The term “BRET” is used to describe a process that is related to FRET,but differs from FRET in that donor is a bioluminescent protein likeluciferase that generates its own luminescence emission in the presenceof a substrate, and that can pass the energy to an acceptor fluorophore.For either BRET or FRET to work, the donor's emission spectrum mustoverlap the acceptor's absorption spectrum, their transition dipolesmust be in an appropriate orientation, and the donor and acceptor mustbe in close proximity (usually within 30-80 Å of each other, dependingon the degree of spectral overlap).

The term “Scintillation Proximity Assay” is used to describe an assaydetermining the distance between two compounds, wherein one compound(bound to a bead) will emit light when radiation from an isotope occursin close proximity and the other compound is containing a radioactiveisotope.

The term “mammalian cell” is intended to indicate any cell of mammalianorigin. The cell may be an established cell line, many of which areavailable from The American Type Culture Collection (ATCC, Virginia,USA) or a primary cell with a limited life span derived from a mammaliantissue, including tissues derived from a transgenic animal, or a newlyestablished immortal cell line derived from a mammalian tissue includingtransgenic tissues, or a hybrid cell or cell line derived by fusingdifferent celltypes of mammalian origin e.g. hybridoma cell lines. Thecells may optionally express one or more non-native gene products, e.g.receptors.

The phrase “fluorescence properties” means absorption properties, suchas wavelength and extension, or spectral properties of the emittedlight, such as wavelength, fluorescence lifetime, intensity orpolarisation, or the intracellular localisation of the fluorophore. Itmay thus be localised to a specific cellular component (e.g. organelle,membrane, cytoskeleton, molecular structure) or it may be evenlydistributed throughout the cell or parts of the cell.

The term “fixed cells” is meant to cover cells treated with acytological fixative such as glutaraldehyde, methanol, acetone orformaldehyde, treatments which serve to chemically cross-link and/orstabilize soluble and insoluble proteins within the structure of thecell or to dehydrate cells. Once in this state, such proteins cannot belost from the structure of the now-dead cell.

The term “cell line” is meant to cover a group of cells, wherein thecells of that group are essentially genetically indistinguishable fromeach other. The cells of a cell line are thus all progeny of the samecell.

The term “comprising” should be understood in an inclusive manner.Hence, by way of example, a composition comprising compound X, maycomprise compound X and optionally additional compounds.

The term “multiple” should be understood as “at least two”.

The term “library of test compounds” should be understood as acollection of test compounds comprising at least 2 different testcompounds.

The term “small organic molecules or compounds” refers herein tonon-oligomeric, carbon containing compounds producible by chemicalsynthesis and generally having a size of less than 600 mass units.

The term “one bead-one compound library” refers to libraries immobilisedon resin beads, wherein each individual resin bead does not comprisemore than one library member in one or multiple copies. In a particularform of such libraries each member is represented by multiple fragmentsof said member obtained by ladder synthesis encoding.

The term “one bead-two compound library” refers to libraries immobilisedon resin beads, wherein each individual resin bead does not comprisemore than one library member in one or multiple copies and wherein eachindividual resin bead in addition to said library member also comprisesan adhesion compound. All beads may comprise identical adhesioncompounds.

DETAILED DESCRIPTION OF THE INVENTION Library of Test Compounds

In the present invention, libraries of compounds are used to screen forcompounds having a desired physiological influence on a living cell. Asused herein, the term “library” means a collection of molecular entitiesor test compounds, herein also designated “library members” obtainedafter a series of chemical transformation.

In preferred embodiments of the present invention the library is acombinatorial library. Non-limiting examples of combinatorial librariesthat may be used with the present invention and methods of producingsuch libraries are given in: Comprehensive Survey of CombinatorialLibrary Synthesis: 1998 Roland E. Dolle and Kingsley H. Nelson, Jr. J.Comb. Chem., 1999, pp 235-282; Comprehensive Survey of CombinatorialLibrary Synthesis: 1999 Roland E. Dolle J. Comb. Chem., 2000, pp383-433; Comprehensive Survey of Combinatorial Library Synthesis: 2000Roland E. Dolle J. Comb. Chem., 2001, pp 477-517; Comprehensive Surveyof Combinatorial Library Synthesis: 2001 Roland E. Dolle J. Comb. Chem.,2002, pp 369-418 and Comprehensive Survey of Combinatorial LibrarySynthesis: 2002 Roland E. Dolle J. Comb. Chem., 2003, pp 693-753. Theskilled person will appreciate that these protocols may be easily beadapted to specific need of a particular embodiment of the presentinvention.

In one embodiment, these molecular entities can be natural oligomers(oligomers of building blocks occurring in Nature) such as peptides,glycopeptides, lipopeptides, nucleic acids (DNA or RNA), oroligosaccharides. By way of example, a natural oligomer may be anypeptide consisting of naturally occurring amino acid, even if saidpeptide comprises a sequence not present in nature. The libraries maycomprise different natural oligomers or the libraries may comprise onlyone kind of natural oligomer, for example the library may be a peptidelibrary. In another embodiment, they can be unnatural oligomers(oligomers comprising one or more building blocks not occurring inNature) such as chemically modified peptides, glycopeptides, nucleicacids (DNA or RNA), or, oligosaccharides, and the like. Said chemicalmodification may for example be the use of unnatural building blocksconnected by the natural bond linking the units (for example, a peptideamide linkage), the use of natural building blocks with modified linkingunits (for example, oligoureas as discussed in Boeijen et al, 2001, J.Org. Chem., 66: 8454-8462; oligosulfonamides as discussed in Monnee etal, 2000, Tetrahedron Lett., 41: 7991-95), or combinations of these (forexample, statine amides as discussed in Dolle et al, 2000, J. Comb.Chem., 2: 716-31.). Preferred unnatural oligomers include oligomerscomprising unnatural building blocks connected to each other by anaturally occurring bond linking. Said oligomers may thus comprise amixture of naturally occurring and unnatural building blocks linked toeach other by naturally occurring bonds. By way of example, the oligomermay comprise naturally occurring amino acids and unnatural buildingblocks linked by peptide bonds f.x. PNA or LNA. Thus, in one embodimentof the invention preferred oligomers comprise modified amino acids oramino acid mimics). Other preferred unnatural oligomers include, forexample oligoureas, poly azatides, aromatic C—C linked oligomers andaromatic C—N linked oligomers. Still other preferred oligomers comprisea mixture of natural and unnatural building blocks and natural andunnatural linking bonds. For example, the unnatural oligomer may be anyof the oligomers mentioned in recent reviews see: Graven et al., 2001,J. Comb. Chem., 3: 441-52; St. Hilaire et al., 2000, Angew. Chem. Int.Ed. Engl., 39: 1162-79; James, 2001, Curr. Opin. Pharmacol., 1: 540-6;Marcaurelle et al., 2002, Curr. Opin. Chem. Biol., 6: 289-96; Breinbaueret al., 2002, Angew. Chem. Int. Ed. Engl., 41: 2879-90. The libraries ofthe invention may also comprise cyclic oligomers, for example cyclicnatural oligomers, such as cyclic peptides or cyclic unnaturaloligomers. In certain embodiments of the invention, libraries of cyclicoligomers may be advantageous to use due to the rigid structure. Thismay result in higher selectively and affinity.

In yet another embodiment, the molecular entities may comprisenon-oligomeric molecules such as peptidomimetics or other small organicmolecules. Peptidomimetics are compounds that mimic the action of apeptidic messenger, such as bicyclic thiazolidine lactam peptidomimeticsof L-proplyl-L-leucyl-glycinamide (Khalil et al, 1999, J. Med. Chem.,42: 2977-87). In a preferred embodiment of the invention, the librarycomprises or even more preferably consists of small organic molecules.Small organic molecules are non-oligomeric compounds of less than about600 mass units containing any of a variety of possible functional groupsand are the product of chemical synthesis, or isolated from nature, orisolated from nature and then chemically modified, and include, forexample, Bayer's urea-based kinase inhibitors (Smith et al., 2001,Bioorg. Med. Chem. Lett., 11: 2775-78). Small organic compounds may forexample be selected from the group consisting of alcohols, ethers,carboxylic acids, aryloxy, acyloxy, thiol, alkylthio, arylthio,heteroarylthio, sulphonyl, sulphoxy, amino, alkylamino, dialkylamino,acylamino, diacylamino, alkoxycarbonylamino, amides, alkyl, branchedalkyl, aryl, heteroaryl, nitro, cyano, halogeno, silyloxy, keto,heterocycles, fused ring systems, fused heterocycles and mixturesthereof, wherein each of the aforementioned may be substitutedindependently on each position with one or more groups selected from thegroup consisting of —H, —OH, —SH, halogen, carboxyl, carbonyl, alkoxy,aryloxy, acyloxy, alkylthio, arylthio, heteroarylthio, sulphonyl,sulphoxy, amino, alkylamino, dialkylamino, acylamino, diacylamino,alkoxycarbonylamino, amides, alkyl, aryl, heteroaryl, nitro, cyano,halogeno, silyloxy, keto, heterocycles, fused ring systems, and fusedheterocycles.

Non-limiting examples of small organic molecule libraries that may beused with the present invention and methods of producing them may forexample be found in the reviews Thompson et al., 1996, Chem. Rev., 96:555-600; Al-Obeidi et al., 1998, Mol. Biotechnol., 9: 205-23; Nefzi etal., 2001, Biopolymers, 60: 212-9; Dolle, 2002, J. Comb. Chem., 4:369-418.

The libraries according to the invention may comprise at least 20, suchas at least 100, for example at least 1000, such as at least 10,000, forexample at least 100,000, such as at least 1,000,000 different testcompounds. Preferably, the libraries comprises in the range of 20 to10⁷, more preferably 50 to 7,000,000, even more preferably 100 to5,000,000, yet more preferably 250 to 2,000,000 different compounds. Ina very preferred embodiment of the present invention the librariescomprises in the range of 1000 to 20,000, such as in the range of 20,000to 200,000 different test compounds. In preferred embodiments of theinvention the library comprises in the range of 10,000 to 1,000,000different test compounds.

Preferably, the libraries to be used with the present invention areimmobilised on resin beads. Said resin beads may be any of the beadsdescribed herein below. At least 2, preferably at least 20, morepreferably at least 100, even more preferably at least 1000, yet morepreferably at least 10,000, for example at least 100,000, such as atleast 1,000,000 resin beads comprising different library members, i.e.different test compounds may be used with the methods according to theinvention. Preferably, the in the range of 20 to 10⁷, more preferably100 to 7,000,000, even more preferably 1000 to 5,000,000, yet morepreferably 5000 to 2,000,000, even more preferably 10,000 to 1,000,000resin beads comprising different library members, are used with themethods according to the invention.

In one very preferred embodiment of the invention, each resin bead doesnot comprise more than one library member in one or more copies, i.e.each resin bead only comprises on kind of test compound, however saidtest compound may be present on the resin bead in multiple copies. Suchlibraries may also be designated one-bead-one-compound libraries.Preferably, each resin beads comprises sufficient copies of said librarymember in order to exert the desired influence of cells attached to saidresin bead and in order to analyse the chemical structure of thecompound. Such libraries may be prepared by different methods, forexample by a split/mix method or by coupling individually a specificcompound to a bead. One-bead-one compound libraries offer the advantagethat once a resin bead has been selected according to the methodsdescribed herein, the desired compound may easily be identified (seeuseful methods herein below).

The libraries may in one preferred embodiment be synthesized directly onresin beads using a split/mix method (vide infra) which gives rise toone-bead-one-compound libraries. Split/mix methods in general comprisethe steps of:

-   1. Providing several pools of resin beads-   2. Performing one or more different chemical synthesis steps on each    pool of resin beads,-   3. Splitting said pools to obtain fractions-   4. Mixing fractions from different pools, thereby obtaining new    pools-   5. Optionally repeating step 1 and 4

Alternatively steps 3 and 4 may be as follows:

-   -   3. Mixing all pools of resin beads, thereby obtaining a mixed        pool    -   4. Splitting the mixed pool of resin beads into reaction        containers thereby obtaining new pools.

One-bead-one-compound libraries may for example be prepared as describedin M. Meldal, Multiple column synthesis of quenched solid-phase boundfluorogenic substrates for characterization of endoprotease specificityin Methods: A Companion to Methods in Enzymology 6:417-424, 1994 or inM. Meldal, The One-bead Two-Compound Assay for Solid Phase Screening ofCombinatorial Libraries in Biopolymers, Peptide Science 66:93-100, 2002;or in Combinatorial peptide library protocols, Ed. by Shmuel Cabilly,Humana Press, 1998, p. 1-24 and 51 to 82.

In another embodiment of the invention the library may be aone-bead-two-compounds library. Each individual resin bead of such alibrary comprises only one library member in one or more copies. Inaddition each individual resin bead comprises a second compound, such asa cell adhesion compound. The cell adhesion compound could for examplebe any of the cell adhesion compounds mentioned herein below. It iscomprised within the invention that several library resin beads, such asall library resin beads comprises identical adhesion compound(s) in oneor more copies. One-bead-two-compound libraries may for example beprepared by a method involving the steps of:

-   1. Providing resin beads comprising a plurality of reactive groups-   2. Reacting said reactive groups with two chemical moeities    comprising different and preferably orthogonal protective groups-   3. Deprotecting a subset of the reactive groups by removal of one    kind of protective groups, preferably selective removal of one kind    of protective group,-   4. Attaching or synthezising a split/mix library of test compounds    to the deprotected reactive group-   5. Deprotecting the remaining reactive groups by removal the other    kind of protective group-   6. Attaching the second compound to the deprotected reactive groups

The method may also be performed by first attaching the second compoundand then synthezising the library. Accordingly, the steps of the methodmay be performed in the following order: 1, 2, 3, 6, 5 and 4. Thelibrary of test compounds may be first synthesized and then attached tothe resin beads or it may be synthesized directly into the resin bead.Similarly, the second compound may be first synthesized and thenattached to the resin beads or it may be synthesized directly into theresin bead.

Preferred resin beads are described in the section “resin beads” hereinbelow. The reactive group may be any suitable reactive group, preferablyhowever, the reactive group is either a hydroxyl group, a thiol or aprimary amino group. The reactive may also preferably be an azido or asecondary amino group. The protective group may be any suitableprotective group known to the person skilled in the art, such as acidlabile, alkaline labile or photolabile protective groups, preferably theprotective group is selected from the group consisting of Fmoc, Boc,Alloc and N₃. It is preferred that the different protective groups maybe removed by different treatment, for example that if one protectivegroup is acid labile, then the other is not acid labile, but instead forexample alkaline labile or photo labile. In an preferred embodiment oneprotective group is Fmoc and the other protective group is Alloc or N₃.Step 3 may for example be performed by a split/mix method as describedherein above, thereby generating a one-bead-one-compound library. Thesecond compound is preferably a cell adhesion compound.

In one embodiment the library may be linked to the resin bead via alinker, which may be a cleavable linker. This may for example beachieved by synthesizing the linker directly on resin beads or couplingthe linker to the resin beads and subsequently coupling or synthesizingthe library onto the resin beads. Thus, before coupling of the librarythe linker preferably comprises a protective group as described hereinabove. The cleavable linker may be any of the cleavable linkersdescribed herein below. If the resin beads are coupled to an adhesioncompound via a cleavable linker it is preferred that the cleavablelinker linking the library is different to the cleavable linker linkingthe adhesion compound. It is in particularly preferred that the linkerare not cleavable by the same mechanism. Thereby, the library may bespecifically released from the resin beads, without release of adhesioncompounds.

In yet another embodiment of the invention the library may be a mixedcompound library, wherein each individual resin bead comprises aplurality of library members.

Selection of an appropriate library is dependent upon the specificembodiment of the invention. For example, a totally random librarydesigned to contain interesting and greatly diverse compounds may beused with the invention. An advantage of this approach is that theoutcome of the screening is not prejudiced in any specific manner. Sincethe invention permits screening of millions of diverse compounds, forexample, immobilized on resin beads, a large number, for example in therange of 3 to 5 million, of random molecules can be used in the ligandlibrary.

Alternatively, a smaller, targeted library (hundreds to thousands ofcompounds) can be used, for example, starting with a known compound orcompounds, and providing numerous variations of these known compoundsfor targeted screening. For example, in embodiments of the inventionwherein compounds modulating the activity of a specific cell surfacemolecule, a compound known to modulate said specific cell surfacemolecule may be used as starting compound for the preparation of atargeted library. Alternatively, a smaller targeted library of compoundsmimicking a compound known to modulate the activity of said cell surfacemolecule may be prepared, for example using computer aided modellingfollowed by chemical synthesis. The smaller, targeted library can alsocomprise random molecules. Examples of libraries and methods ofpreparing such libraries, which may useful in embodiments of theinvention, wherein the cellular response is mediated through a G-proteincoupled receptor are described in C. Haskell-Luevano, A. Rosenquist, A.Souers, K. C. Khong, J. A. Ellman, and R. D. Cone, 1999, J. Med. Chem.42:4380-4387. Compounds that activate the mouse melanocortin-1 receptoridentified by screening a small molecule library based upon the b-turn.J. Med. Chem. 42:4380-4387, 1999; A. J. Souers, A. A. Virgilio, A.Rosenquist, W. Fenuik, and J. A. Ellman. Identification of a potentheterocyclic ligand to somatostatin receptor subtype 5 by the synthesisand screening of b-turn mimetic libraries. J. Am. Chem. Soc. 121(9):1817-1825, 1999; J. Bondebjerg, Z. Xiang, R. M. Bauzo, C.Haskell-Luevano, and M. Meldal. A solid phase approach to mousemelanocortin receptor agonists derived from a novel thio-ether cyclizedpeptidomimetic scaffold. J. Am. Chem. Soc. 124:11046-11055, 2002; B. A.Harrison, G. W. Pasternak, and G. L. Verdine. 2,6-dimethyltyrosineanalogues of a stereodiversified ligand library: highly potent,selective, non-peptidic m opioid receptor agonists. J. Med. Chem.46:677-680, 2003; G. R. Marshall. Peptide interactions with G-proteincoupled receptors. Peptide Science 60:246-277, 2003; P. N. Arasasingham,C. Fotsch, X. Ouyang, M. H. Norman, M. G. Kelly, K. L. Stark, B. Karbon,C. Hale, J. W. Baumgartner, M. Zambrano, J. Cheetham, N. A. Tamayo, andStructure-Activity relationship of (1-aryl-2-piperazinylethyl)piperazines: Antagonists for the AGRP/Melanocortin receptor binding. J.Med. Chem. 46:9-11, 2003. Further useful libraries are described inexamples 4, 5 and 6 herein below: The person skilled in the art willappreciate that other libraries may be prepared by adapting theprotocols described in the aforementioned references. The library maycontain a parallel array of random modifications of one or more testcompounds. In one embodiment, the library may be formed as a parallelarray of random modifications to a known compound or compounds. The term“parallel array” is meant to cover synthesis of a library by subjectinga given compound to a known set of reactions in an isolated vessel orwell. Thus, the nature of a compound in a given container or well isknown. The array of test compounds is preferably prepared directly onresin beads using techniques known by those skilled in the art. Briefly,the resin may be portioned into a number of vessels or wells, usuallyless than 500 and the reagents added. There is in general no mixing stepand after the appropriate washing steps, subsequent reactions arecarried out by addition of additional reagents to the wells. There is noexponential increase in the number of compounds generated and that isequal to the number of vessels used. The compound can be easilyidentified by keeping track of the reagent added to each well.

The library may also have been prepared by parallel synthesis using atag to enable identification of, what chemical synthesis steps theindividual resin bead has been submitted to. This may for example bedone by IRORI or radiofrequency tag. Alternatively, chemical synthesissteps may be performed in parallel to preparing a polymeric tag.Identification of the tag will thus provide knowledge of the compound.

Attachment of a label to a compound may alter the properties of saidcompound. Hence, in one embodiment of the present invention, thecompounds of the library are not labelled, i.e. the compounds are notconnected to a detectable label, such as a fluorescent component, anucleic acid or a nucleic acid homologue such as PNA, a dye, a probecomprising a reactive moiety or the like. In particular it is preferredthat all compounds are not connected to the same detectable label.

In one aspect the present invention also relates to methods ofsynthezising libraries of test compounds, wherein said libraries are inparticular useful for the screening methods of the invention.

In one embodiment, the invention thus relates to methods of synthesisinga cyclic peptide or peptide mimetic library, comprising the steps

-   -   i) Providing a plurality of peptides or peptide mimetics,        (preferably peptides) covalently linked to an azide moiety and        an acetylene moeity; and    -   ii) cyclizing said peptide or peptide mimetic through a Cu(I)        catalysed reaction between said azide- and said acetylene        moiety; and    -   iii) thereby obtaining a library of cyclic peptides or peptide        mimetics.

Each peptide preferably only comprises one azide moeity and oneacetylene moiety. An example of a method of preparing such a library isgiven in example 4 herein below.

In another embodiment, the invention relates to methods of synthesisinga library of heterocyclic ureas, comprising the steps of

-   -   i) Providing a plurality of urea containing peptide aldehydes;        and    -   ii) Subjecting said urea containing peptides to an        intramolecular Pictet-Spengler reaction; and    -   iii) Thereby obtaining a library of heterocyclic ureas

Said urea containing peptide aldehydes are preferably peptidescovalently linked to at least one urea moeity and one aldehyde moeity.The intramolecular Pictet-Spengler reaction may for example be performedas described in WO2004/113362 claiming priority from Danish patentapplication PA 2003 00967, both are hereby incorporated by reference.

An example of a method of preparing such libraries is given in examples5 and 5a herein below.

The peptides used for preparation of any of the libraries mentionedabove may be oligomers of naturally occurring or not naturally occurringamino acids or a mixture of both, preferably they are oligomers of the20 amino acids naturally present in proteins, wherein said amino acidsmay be in either D- or L-form. It is preferred that each peptide (orpeptide mimetic) is immobilised on a solid support, such as any of thesolid supports mentioned herein below. More preferably the solid supportis resin beads and it is preferred that each resin bead comprises onlyone library member in one or more copies.

Preferably at least 2, such as at least 10, for example at least 100,such as at least 1000, for example at least 10,000 different peptidesand/or peptide mimetics are provided. Each peptide may comprise in therange of 2 to 100 amino acids, such as in the range of 2 to 50 aminoacids, for example 2 to 25 amino acids, such as in the range of 2 to 15amino acids, for example 2 to 10 amino acids, such as in the range of 3to 8 amino acids, for example 4 to 6 amino acids,

The invention also relates to libraries prepared by any of the methodsdescribed above.

Libraries of heterocyclic compounds obtained by cyclisation of a peptidealdehyde through an intramolecular Pictet-Spengler reaction may also beused with the present invention. Such libraries may for example be anyof the libraries described in WO2004/113362 claiming priority fromDanish patent application PA 2003 00967, both are hereby incorporated byreference.

Resin Beads

The library members of this invention are preferably bound to a solidsupport. Preferred solid supports to be used with the present inventionare resin beads (see herein below).

The solid support may however also be a spot or region on a surface or aplated gel or a membrane. A spot or a region is a defined area on saidsurface, to which the library member is covalently bound. One cantherefore envisage one surface comprising a plurality of spots orregions, wherein each such spot or region is covalently attached to onlyone library member in one or more copies. Said surface could for examplebe a silicium wafer, a glass surface, a plastic surface or a gel.Plastic surface may for example be prepared from polystyrene,polycarbonate poly-propylene, ethylene and/or teflon. Gels could beprepared from for example poly acrylamid or PEGA.

In this invention however, the compounds of the library are preferablybound to a resin bead, conferring the advantage of compartmentalized“mini-reaction vessels” for attachment of cells.

In general more compounds may be screened and several of the steps inthe procedure may be performed on one bead with sufficient material.Hence, preferably, the library is bound to resin beads. Each member ofthe library is a unique compound and is physically separated in spacefrom the other compounds in the library, preferably, by immobilizing thelibrary on resin beads, wherein each bead at the most comprises onemember of the library. Depending on the mode of library synthesis, eachlibrary member may contain, in addition, fragments of the librarymember. Since ease and speed are important features of this processinvention, it is preferred that the screening step take place on thesame solid support used for synthesis of the library, and also thatidentification of the members of the binding pair can take place on thesame support, such as on a single resin bead. Thus, preferred solidsupports useful in the process invention satisfy the criteria of notonly being suitable for organic synthesis, but are also suitable forscreening procedures, such as “on-bead” screening as well as suitablefor attachment of cells. It is furthermore preferred that the resin beadis suitable for “on-bead” identification of library members as describedherein below. The resin bead may be prepared from any suitable materialsuch as polystyrene, polyethylene polyacrylamide, controlled pore glassor PEG. The resin bead could thus for example be selected from the groupconsisting of Toyopearl, sepharose, sephadex, CPG, silica, POPOP, PEGA,SPOCC, Expansin, Tentagel, Argogel, Polystyrene, Jandagel,polydimethylacrylamide resin, Poly-acrylamide resin, kieselghursupported resins and polystyrene supported resins. Hydrophilic supportsare preferred. Examples of preferred hydrophilic resin beads includesTentaGel (commercially available from Rapp polymere, Tübingen, Germany),ArgoGel (commercially available from Argonaut Technologies Inc., SanCarlos, Calif.), PEGA (commercially available from VersaMatrix,Copenhagen), POEPOP (Renil et al., 1996, Tetrahedron Lett., 37: 6185-88;available from Versamatrix, Copenhagen, Denmark) and SPOCC (Rademann etal, 1999, J. Am. Chem. Soc., 121: 5459-66; available from Versamatrix,Copenhagen, Denmark). Examples of on-bead screening attempts aredescribed in the following references: Chen et al., 1996, MethodsEnzymol., 267: 211-19; Leon et al., 1998, Bioorg. Med. Chem. Lett., 8:2997-3002; St. Hilaire et al., 1999, J. Comb. Chem., 1: 509-23; Smith etal., 1999, J. Comb. Chem., 1: 326-32; Graven et al., 2001, J. Comb.Chem. 3: 441-52; Park et al., 2002, Lett. Peptide Sci., 8: 171-78).TentaGel and ArgoGel are made up of polyethylene glycol chains graftedon to a polystyrene core. However, use of these supports in biologicalscreening is limited by a size restriction, and by denaturation ofcertain proteins, particularly enzymes.

Preferred resin beads according to the present invention are resinbeads, useful for on-bead library synthesis, screening andidentification of ligand/protein. Hence, preferred resins according tothe present invention are resin comprising polyethylene glycol. Morepreferably, the resin is PolyEthyleneGlycol Acrylamide copolymer (PEGA),Super Permeable Organic Combinatorial Chemistry (SPOCC) orPoly-OxyEthylene-PolyOxyPropylene (POEPOP) resin. Another preferredresin comprises a crosslinked polyacrylamide resin.

PEGA (PolyEthyleneGlycol Acrylamide copolymer; Meldal M., 1992,Tetrahedron Lett., 33: 3077-80), POEPOP(PolyOxyEthylene-PolyOxyPropylene; Renil et al., 1996, TetrahedronLett., 37: 6185-88) and SPOCC (Super Permeable Organic CombinatorialChemistry; Rademann et al, 1999, J. Am. Chem. Soc., 121: 5459-66) resinsare made primarily of polyethylene glycol and swell well in organic aswell as aqueous solvents. Because they have very reduced or nonon-specific binding, PEGA and SPOCC resins have been effectively usedin the screening of myriad proteins including enzymes of differentclasses. Furthermore, these resins are available in different pore sizesand can allow large proteins to enter while retaining activity. Forexample, PEGA6000 resins allow proteins up to 600 kDa to enter. In theExamples below, PEGA4000 and PEGA1900 resin with a molecular weight cutoff of 200 and 90 kDa, respectively, are used for screening. Inprinciple, any hydrophilic support that is useful for compartmentalizedsynthesis, retains the activity of the proteins, and has minimalnon-specific binding, may be used in this process invention.

One aspect of the invention relates to a method comprising the step ofproviding multiple resin beads capable of supporting growth of cells.Preferably, all resin beads provided are capable of supporting growth ofcells. In one preferred embodiment all resin beads are similar and eachis capable of supporting growth of cells, wherein the resin beads onlydiffers by comprising different library members. In embodiments of theinvention wherein the resin beads comprise a cell adhesion molecule, itis preferred that at least 10%, more preferably at least 20%, even morepreferably at least 30%, yet more preferably at least 40%, even morepreferably at least 50%, yet more preferably at least 60%, %, even morepreferably at least 70%, yet more preferably at least 90%, even morepreferably essentially all, yet more preferably all resin beads comprisethe cell adhesion molecule as well as a library member.

Cells

The cells to be used with the present invention may be any useful cellsavailable or prepared for the purpose. Preferably, the cells areselected from the group consisting of mammalian cells. For example thecells may be human cells. The cells may be cells capable of growing insuspension or they may be adherent cells. Adherent cells may preferablybe cultivated directly on the resin beads used with the invention (seealso herein below). It is preferred that the cells are adherent cells.Cells with a better adherence are preferred over cells with a pooreradherence. Cells which adhere well to resin beads comprising an adhesioncompound as described herein above are very preferred.

Cells could for example be primary cells or established cell lines.Preferred cell lines include but are not limited to those mentioned inTable 1.

TABLE 1 Cell line Species Tissue Morphology 3T3-L1 Mouse Embryonicfibroblast Fibroblast 3T3-Swiss Mouse Embryo Fibroblast albino (CCL-92)A10 Rat thoracic aorta Myoblast Att 20 Mouse Pituitary Small round cellsBAE Cow Aorta Endothelial Balb/c Mouse Embryonic fibroblast FibroblastBHK:R P.1#4aa PTP1B fl BHK-21 Hamster Kidney Fibroblast BHK467 HamsterKidney BHK570 Hamster Kidney Fibroblast BJ Human Foreskin FibroblastC2C12 Mouse Muscle Myoblast Caki-1 Human Kidney Epithelial CAL-54 HumanKidney Epithelial CHOhIR Chinese Ovary Fibroblast hamster CHO-K1 HamsterOvary Epithelial COS 1 Monkey Kidney Fibroblast COS 7 Monkey KidneyFibroblast G-8 Mouse Muscle Myoblast GT1-7 HCT 116 Human ColorectalEpithelial HEK293 Human Embryonic kidney Epithelial Hela Human Cervixadenocarcinoma Epithelial HEP-G2 Human Liver Epithelial HT-1080 HumanFibrosarcoma Epithelial HT-29 Human Colon Epithelial HUVEC HumanUmbilical vein Endothelial Ins-1 Jurkat clone E6-1 Human T lymphocyteLymphoblastoid K-562 Human Bone marrov Lymphoblastoid L-6 Rat MuscleMyoblast MCF 7 Human Mammary Gland Epithelial MDA-MB-231 HumanAdenocarcinoma Epithelial MDA-MB-468 Human Mammary Gland Epithelial MDCKCanine Kidney Epithelial Min 6 Mv 1 Lu (NBL-7) Mink Lung EpithelialNIH-3T3 Mouse Embryo Fibroblast PAE Pig Aorta PC 12 Rat Adrenal glandPC-3 Human Prostate Epithelial RAT2 Rat Normal Fibroblast RAW 264.7Mouse Monocyte RIN Rat Epithelial SK-ML-28 Human Melanoma SK-N-AS HumanNeuroblastoma Epithelial SK-N-DZ Human Neuroblastoma Epithelial SK-N-F1Human Brain Epithelial SK-NM-C Human Neuroepithelioma Epithelial SK-N-SHHuman Caucasian neuroblastoma Epithelial SW480 Human ColorectalEpithelial U-2 OS Human Bone, osteosarcoma Epithelial U-87 MG HumanBrain Epithelial U937 Human Lymphoma Monocyte VERO Monkey KidneyFibroblast-like WI-38 Human Lung Fibroblast WM-266-4 Human SkinEpithelial WEHI Human

In one embodiment of the invention the cells have been genetically orotherwise modified in order to enhance their usability with the presentinvention. The modification may be stable or only transient or a mixtureof both. For example, the cells may have been modified to contain one ormore of the reporter systems described herein below. Depending on thenature of the reporter system this may be achieved by a number ofdifferent methods. For example, if the reporter system comprises anucleic acid, said nucleic acid may be inserted into said cell byconventional recombinant techniques (see below).

In another preferred example the cell comprises a nucleic acidcomprising a first nucleotide sequence encoding a cell surface moleculeoperably linked to a second nucleotide sequence not naturally associatedtherewith directing expression of said first sequence. The cell surfacemolecule may be any of the cell surface molecules described hereinbelow. Such cells are in particular useful for identification ofcompounds modulating the activity of said cell surface molecule. Saidnucleic acid may be introduced transiently or stably into said cells.

Useful second sequences includes for example promoters active in theparticular cells, for example mammalian promoters, viral promoters orsynthetic promoters. A large number of useful eukaryotic promoters areknown to the person skilled in the art, useful promoters are for exampledescribed in“Mechanism of Transcription” (1998) Cold Spring HarborSymposia on Quantitative Biology Vol. LXIII; Cold Spring HarborLaboratory Press

Such promoters may be constitutively active or they may be active onlytemporarily. In one example the promoter may be regulated by an externalsignal, for example the promoter may be inducible or repressable.

The nucleic acid may be inserted into the cells by any useful method,for example by conventional recombinant techniques, such as any of thetechniques described in Sambrook et al., Molecular Cloning: A LaboratoryManual, 1989, Cold Spring Harbor Laboratory, New York, USA

In another embodiment the cells are primary cells. Primary cells arecells with a limited life span that preferably are derived from amammalian tissue. Preferred primary cells are cells which are adherent.The mammalian tissue may for example be a human tissue, such as healthyor diseased tissue. In one embodiment the tissue is or comprises aneoplastic tissue, for example tissue removed from a cancer patient bysurgery, for example from a patient suffering from melanoma, breastcancer or colon cancer. The tissue may also be hypertrophic cells, suchas cardiac myocytes. Preferably said cancer patient has not beensubjected to radiotherapy prior to surgery. In embodiments of theinvention wherein the cells are primary cells it is preferred that thereporter system is endogenous to said primary cells.

Cell Attachment to Resin Beads and Cell Cultivation

The present invention relates to methods comprising the step ofattaching cells comprising a reporter system(s) to resin beads. Thecells may for example attach to said resin beads directly or byattaching a second compound conferring adhesion to the resin bead.

The resin beads useful for the present invention should preferably beable to support cell growth. The resin beads may per se be able tosupport cell growth, however frequently the resin beads will comprise acell adhesion compound that enables the resin beads to support growth ofcells. Said cell adhesion compound may be coupled to said resin beads byany useful means known to the person skilled in the art depending on thenature of the cell adhesion compound.

Any cell adhesion compound known to the person skilled in the art may beused with the present invention. It is frequently an advantage if thecell adhesion compound comprises at least one positively charged moietyat neutral pH, more preferably the cell adhesion compound has a positiveoverall netcharge at neutral pH.

In one preferred embodiment of the invention the cell adhesion compoundcomprises a peptide or a polypeptide, more preferably the cell adhesioncompound consists of a peptide. Such peptides are herein also designated“adhesion peptides”.

Said peptide preferably consists of in the range of 4 to 100, preferablyin the range of 4 to 75, more preferably in the range of 4 to 50, evenmore preferably in the range of 4 to 30, yet more preferably in therange of 4 to 25, even more preferably in the range of 4 to 20, yet morepreferably in the range of 4 to 15, such as in the range of 4 to 10, forexample in the range of 4 to 8, for example in the range of 6 to 7 aminoacids. In general, it is sufficient if the peptide comprises at least 4amino acids.

It is preferred that the peptide comprises at least one amino acidselected from the group consisting of arginine and lysine, morepreferably the peptide comprises at least 2 basic amino acids, such as 3basic amino acids selected from the group consisting of Arg and Lys,even more preferably the peptide has an overall positive netcharge. Inone preferred embodiment the peptide comprises the following sequence of4 amino acids: basic-basic-lipophilic-basic. Basic amino acids may forexample be selected from the group consisting of arginine and lysine,whereas the lipophilic amino acid may be selected from the groupconsisting of Gly, Ala, Val, Leu, Ile, Phe, Trp, Pro and Met of either Dor L-form. Preferably, the peptide comprise at least 1, preferably atleast 2, more preferably at least 3, even more preferably at least 4amino acid on the D-form, yet more preferably all amino acids are on theD-form. Preferably D-amino acids are used to enhance the metabolicstability but also L-amino acids may be used.

Preferred examples of peptides useful as cell adhesion compounds aregiven in table 2 herein below:

TABLE 2 No 1 2 3 4 5 6 7 SEQ ID NO  1 ala arg ile arg ile gln hisSEQ ID: 1  2 ala lys cys arg trp cys met SEQ ID: 2  3 ala lys ala argcys lys ser SEQ ID: 3  4 ala lys tyr trp ser tyr lys SEQ ID: 4  5 alahis leu tyr arg asn lys SEQ ID: 5  6 ala arg arg cys phe arg aspSEQ ID: 6  7 ala ala arg his cys tyr tyr SEQ ID: 7  8 ala tyr tyr cysgln gln arg SEQ ID: 8  9 ala asp leu lys arg pro met SEQ ID: 9 10 alagly gly lys arg lys phe SEQ ID: 10 11 ala pro arg lys arg cys glySEQ ID: 11 12 ala thr arg arg val ala arg SEQ ID: 12 13 ala gly lys lysasn lys asn SEQ ID: 13 14 ala ala lys arg trp lys phe SEQ ID: 14 15 alaarg trp pro tyr arg gly SEQ ID: 15 16 ala leu tyr trp thr trp argSEQ ID: 16 17 ala ala tyr arg trp tyr arg SEQ ID: 17 18 ala arg cys ilearg gly asp SEQ ID: 18 19 ala thr lys cys lys gly arg SEQ ID: 19 20 alaval tyr met arg asn ile SEQ ID: 20 21 ala arg lys arg ile arg glnSEQ ID: 21 22 ala lys ile arg glu lys arg SEQ ID: 22 23 ala arg arg phelys met tyr SEQ ID. 23 24 arg arg phe lys SEQ ID: 24 25 arg arg ile argSEQ ID: 25 26 leu arg his arg leu lys SEQ ID: 26 27 lys phe gly gln lysSEQ ID: 27 28 lys val tyr met his lys SEQ ID. 28 29 ile arg tyr arg leuarg SEQ ID: 29 30 ala gln arg pro arg trp SEQ ID: 30 trp tyr ala lys argarg SEQ ID: 31 lys arg ile arg gln arg leu arg SEQ ID: 32 lys arg ilearg gln arg lys SEQ ID: 33 arg ile arg gln arg SEQ ID: 34 arg gln argile arg SEQ ID: 35 lys phe gly gln lys cys SEQ ID: 36 arg arg leu leupro ile SEQ ID: 37 pro phe arg lys lys cys SEQ ID: 38 tyr arg trp argile ala SEQ ID: 39 arg ser lys arg ile asn SEQ ID: 40 arg ser ala lysarg cys SEQ ID: 41 lys lys gln phe trp phe SEQ ID: 42 arg met lys leuhis lys SEQ ID: 43 arg his trp gly arg ile SEQ ID: 44 thr lys arg leulys thr SEQ ID: 45 thr lys gly lys ala lys SEQ ID: 46 ala lys thr arghis arg SEQ ID: 47 asn arg pro arg val arg SEQ ID: 48 val pro arg lysval gln SEQ ID: 49 lys met arg tyr cys gln SEQ ID: 50 ile arg lys hisleu ile SEQ ID: 51 pro arg arg val val ile SEQ ID: 52 lys arg glu serlys arg SEQ ID: 53 ser arg lys asp arg lys SEQ ID: 54 arg cys lys lysleu ile SEQ ID: 55 arg lys leu arg val asn SEQ ID: 56 val arg thr valarg val SEQ ID: 57 arg ala phe lys tyr tyr SEQ ID: 58 ile thr arg argthr gln SEQ ID: 59 lys met pro lys lys asn SEQ ID: 60 lys pro lys metmet cys SEQ ID: 61 lys lys met arg phe trp SEQ ID: 62 lys lys lys phetyr tyr SEQ ID: 63 lys ser asn lys val arg SEQ ID: 64 lys trp pro hishis arg SEQ ID: 65 arg his ile gln trp tyr SEQ ID: 66 leu arg leu lyspro lys SEQ ID: 67 glu arg lys arg cys thr SEQ ID: 68 arg arg ala arggln asp SEQ ID: 69 arg glu lys gly ala arg SEQ ID: 70

Furthermore, preferred peptide may be any of the peptides identified byany of SEQ ID: 1 to 70, preferably any of SEQ ID: 1 to 23 and 26 to 35,such as SEQ ID: 1 to 23, for example SEQ ID: 25 to 35, wherein 3 aminoacids, preferably 2 amino acids, more preferably 1 amino acid have beensubstituted for another amino acid. Preferably, said substitution is aconservative substitution, i.e. substitution for an amino acid withsimilar characteristics. Said characteristic could for example beacidic/basic properties, polarity or lipophilicity. It is also comprisedwithin the invention that the peptide may be a peptide of abovementioned size comprising any of the peptides identified by SEQ ID: 1 to70. In particular, in order to immoblised the peptide on a resin bead itmay be useful to synthesise the adhesion peptide on an amino acidimmobilized on the resin bead, for example a Gly.

In one embodiment the peptide is preferably selected from the groupconsisting of peptides identified by SEQ ID: 21 to 23 and 36 to 35, morepreferably from the group consisting of 26 to 35, even more preferablySEQ ID:35. In another embodiment the peptide defined by SEQ ID:21 ispreferred.

In one embodiment of the invention it is preferred that the peptide haslow or essentially no fluorescent properties. It is particularlypreferred that the peptide has low or essentially no fluorescentproperties when attached to a solid support, such as a resin bead. By“essentially no fluorescent properties” is meant that the peptide doesnot emit any detectable fluorescence. This is in particularly relevantfor embodiments of the invention wherein the detectable output isfluorescence (see herein below). Preferred peptides to use with thisembodiment of the invention may be selected from the group consisting ofSEQ ID:26 to 35.

Peptides useful as cell adhesion compounds may be identified using anysuitable method. Said method may for example include the steps of

-   -   i) coupling a test peptide to a resin bead;    -   ii) incubating said resin bead with cells under cell cultivation        conditions;    -   iii) testing whether said cells attach to said resin bead    -   iv) identification of the peptide sequence        wherein the test peptide is useful as cell adhesion compound If        more cells attach to said resin bead in the presence, than in        the absence of said test peptide. Preferably, the test peptide        is useful as cell adhesion compound If at least 200, more        preferably at least 500, even more preferably at least 1000        cells attach to said resin bead after incubation. This is in        particular the case in embodiments of the invention, wherein the        resin beads are PEGA beads. For example useful test peptides may        be identified as described in example 1 herein below.

In embodiments of the invention wherein it is preferred that the peptidehas no or low fluorescence it is preferred that the method comprises anadditional step performed at any point subsequent to step i), such asimmediately subsequent to step i) prior to step ii). Said additionalstep comprises testing whether said peptide has fluorescent properties.This may for example be performed by sorting resin beads in a FABS ormanually with the aid of a fluorescence microscope. If this is doneprior to step ii) then only resin beads with no or low fluorescenceproperties are incubated with cells, A non-limiting example of a usefulmethod is described in example 1a.

The peptide may be coupled to the resin bead by any useful method, forexample by synthesising the peptide directly onto an aminofunctionalised resin bead using a standard Fmoc-protocol for peptidesynthesis. Other protective groups may be used instead of Fmoc, forexample N₃ or Alloc. In one embodiment Alloc is the preferred protectivegroup. It is preferred that different protecting group are used forsynthesis of the adhesion peptide or for library synthesis. The peptidemay also be synthesised by anchoring an Fmoc amino acid to a hydroxylfunctionalised resin bead, such as a hydroxymethylbenzoic acidderivatised PEGA resin followed by peptide assembly using standard Fmoctechnology as described in B. Blankemeyer-Menge, M. Nimtz, and R. Frank,An Efficient method for anchoring Fmoc-amino acids tohydroxyl-functionalised solid supports. Tetrahedron Lett. 31:1701-1704,1990. Sidechains may be protected with acid labile protecting groupssuch as t-Bu, Trt, Pmc, Boc etc. The protected peptide may for examplebe cleaved off the resin using alkaline conditions or hydrazine and thestructure may be determined e.g. by on bead Edman Degradtion. Annon-limiting example of a method for synthesizing an adhesion peptide isgiven in example 5a, “Synthesis of adhesion peptide” herein below.

In one embodiment the adhesion compound may be linked to the resin beadvia a linker, which may be a cleavable linker. This may for example beachieved by synthesizing the linker directly on resin beads or couplingthe linker to the resin beads and subsequently coupling or synthesizingthe library onto the resin beads. Thus, before coupling of the librarythe linker preferably comprises a protective group as described hereinabove. The cleavable linker may be any of the cleavable linkersdescribed herein below. If the resin beads are coupled to the libraryvia a cleavable linker it is preferred that the cleavable linker linkingthe adhesion compound is differentially cleavable.

In embodiments wherein cells adhere to the resin bead via the adhesioncompound and the adhesion compound is attached to the resin bead via acleavable linker, cells may be at least partially or even essentiallyfully released from the resin bead by cleavage of the cleavable linker.

Testing whether cells attach to resin beads may be done by anyconventional methods, such as by manual inspection with the aid of alight microscope. If the cells have fluorescent properties, for exampleif the cells express a fluorescent protein, then resin beads withattached cells may be identified using a fluorescent microscope or aFABS, preferably a fluorescent microscope.

In one preferred embodiment of the invention, the cells may becultivated directly on the resin beads. In general, a method ofcultivating cells on resin beads may comprise the steps of

-   -   Providing resin beads capable of supporting growth of cells    -   Seeding cells onto said resin bead    -   Incubating said resin beads comprising said cells in a cell        culture medium under cell cultivation conditions    -   Optionally allowing said cells to divide on said resin bead    -   Thereby cultivating cells on resin beads

The cells may adhere actively to the resin beads and will then generallybe referred to as adherent cells.

Cells cultivation conditions depends on the specific cells. For a largenumber of mammalian cells, such conditions comprise high humidity,preferably close to 100%, approximately 5% CO₂ and around 37° C. It isoften desirable to keep the resin beads immersed in a suitablecultivation medium and frequently it is also desirable that the resinbeads can be circulated within said medium, for example by stirring orrotation. Said stirring or rotation may be continuous or in intervals.It is also possible the container comprising the resin beads is simplyrocked gently a few times every now and then.

In another embodiment of the invention cells may be attached to resinbeads, without active adherence. For example, this may be the case fornon-adherent cells, i.e. cells that may be cultivated in suspension.

In one embodiment of the invention more than one cell line or type ofprimary cell is attached to or cultivated on the beads. Hence forexample 2, such as 3, for example 4, such as 5, for example 6, such as7, for example 8, such as 9, for example 10, such as in the range of 10to 20, for example in the range of 20 to 50, such as more than 50different cell lines may be attached to or cultivated on said beads.Also different specific primary cells may be attached to the cultivatedbeads.

It is possible that a subgroup of resin beads only comprise one cellline or a specific kind of primary cells and another subgroup of resinbeads comprises another cell line or another specific kind of primarycell and so forth. However, it is also possible that in principle everyresin beads comprises all the different cell lines.

Intermediates between these two extremes may also be envisaged.Preferably, said different cell lines and/or primary cells comprisedifferent reporter systems, hence it is possible that the different celllines are derived from the same parent cell lined by insertion ofdifferent reporter systems. However, the different cell lines may alsobe unrelated.

Cleavable Linkers

The library of test compounds and/or the adhesion compound may in oneembodiment be linked to the resin beads or solid supports by a cleavablelinker.

The cleavable linker may be any chemical moiety which may be used toattach a molecule to a solid support either covalently or via complexformation, and thereafter is capable of releasing said molecule by theaction of either acid, base, electrophiles, nucleophiles, oxidativeagents, reductive agents, metals or light. Preferably, the cleavablelinker attaches the library member/adhesion molecule to the solidsupport covalently. A comprehensive review describing state of the artfor “cleavable linkers” is “Linkers and Cleavage Strategies inSolid-Phase Organic Synthesis and Combinatorial Chemistry”, F. Guillier,D. Orain, and M. Bradley, Chem. Rev. 2000, 100, 2091-2157. Any of thecleavable linkers described therein may be used with the presentinvention.

Examples of useful acid labile linkers include the most commonly usedlinkers for acidic detachment from a solid support, the Wang and Rinklinkers. Examples of useful base-labile linkers includes Wang and HMBAlinkers, which may be cleaved under alkaline conditions. Light sensitivecleavable linkers are linkers which, upon the action of light with agiven wave length and intensity, may release the library member/adhesioncompound from the solid support. Photo-labile linkers cleavable byirradiation with UV-light may be o-nitrobenzyl type of linkers (nitratedanalogs of the Wang linker), NBA type linkers or Holmes-type linkers.Paladium linkers may also be used with the invention.

In one embodiment photolabile linkers are preferred

Cell Surface Molecules

In one particularly preferred embodiment of the invention the methods ofthe invention involve identification of compounds modulating a cellularresponse, which is mediated through a cell surface molecule. Hence, theinvention, for example may be useful for identifying compoundsmodulating the activity of a cell surface molecule, preferably a cellsurface molecule capable of activating/repressing a signal transductionpathway. Within the context of the present invention the term “signaltransduction pathway” should be understood in its common cell biologicalmeaning, i.e. modulation of an intracellular event triggered by a cellsurface receptor.

Signal transduction pathways may for example involve steps ofphosphorylation, cleavage of proteins, synthesis of cAMP, activation oftranscription, inhibition of transcription, change i intracellular Ca²⁺concentration, change in membrane potential, subcellular relocalisationof cellular components, complex formation of cellular components,degradation of cellular components and/or change in energy metabolism

The cell surface molecule is preferably a protein, more preferably aprotein that is accessible from the extracellular surface. Yet morepreferably, the cell surface molecule is a cell surface protein receptor(herein also merely designated “receptor”). A “receptor” within themeaning of the present invention, is a molecule, which at leastsometimes is localised at the cell surface and which is capable orassociating with at least one ligand. The ligand binding site isaccessible from the extracellular surface. Frequently, association withsaid ligand may alter the activity of the receptor.

In a preferred embodiment the cell surface molecule is a G-proteincoupled receptor (GPCR). GPCR is a family of receptors coupled to atrimeric G-protein. GPCR to be used with the invention preferably have 7transmembrane domains. Examples of useful GPCR are given in table 3.

GPCR may be divided into subfamilies, accordingly the GPCR may selectedfrom the group consisting of GPCR belonging the rhodopsin like family,the secretin family or the metabotropic family, preferably from thegroup consisting of GPCR belonging the rhodopsin like family or thesecretin family.

Rhodopsin like GPCR are also referred to as Class I GPCR. They arecharaterised by a structurally similarity to the Rhodopsin receptor.Preferred examples of members of this family includes receptors for thefollowing ligands: Acetylcholine (muscarinic & nicotinic),Adrenoceptors, Alpha Adrenoceptors, Beta Adrenoceptors, Dopamine,Histamine, Serotonin (5-HT), Angiotensin, Bradykinin, C5a anaphylatoxin,Fmet-leu-phe, Interleukin-8, ochernokine, Orexin, Nociceptin, CCK(Gastrin), Endothelin, Melanocortin including any of melanocortin 1 to 5receptors, Neuropeptide Y, Neurotensin, Opioid, Somatostatin, Tachykinin(Substance P, NKA₁), Thrombin, vasopressin-like, Galanin, Folliclestimulating hormone, Lutropinchoriogonadotropic, Thyrotropin, Rhodopsin,Opsin, Prostaglandin, Lysophosphatidic Acid, Sphingosine-1-phosphate,Leukotriene, Prostacyclin, Thromboxane, Adenosine, Purinoceptors,Cannabis, Platelet activating factor, Gonadotropin-releasing Hormone,Thyrotropin-releasing hormone, Growth hormone-inhibiting factor orMelatonin.

Secretin like GPCR are also referred to as Class II GPCR. They arecharaterised by a structurally similarity to the Secretin receptor.(Accession No NM_(—)002980) Preferred examples of members of this familyincludes receptors for the following ligands: Secretin, calcitonin,Corticotropin releasing factor/urocortin, Gastric inhibitory peptide(GIP), Glucagon, Glucagon-like Peptide 1 (GLP-1), Growthhormone-releasing hormone, Parathyroid hormone, PACAP or Vasoactiveintestinal polypeptide (VIP).

Metabotropic GCPR are also referred to as class III GPCR. Preferredexamples of members of this group includes receptors for the followingligands: Metabotropic Glutamate, GABA₈, or Extracellular CalciumSensing.

In another preferred embodiment of the invention the GPCR is coupled toa G-protein, such as G_(S), that stimulates adenylate cyclase. In yetanother preferred embodiment of the invention the GPCR is coupled to aG-protein, such as G_(I), that inhibits adenylate cyclase. Examples ofGPCRs coupled to G_(S) or G_(I) are given in table 3.

Gene Ontology Blast Serverfull

TABLE 3 Gene symbol* Gene Ontology Blast Server Full name G-proteinsignaling, coupled to cyclic nucleotide second messenger NEUY_HUMANNeuropeptide Y precursor [Contains: Neuropeptide Y ACM2_HUMAN Muscarinicacetylcholine receptor M2 SY02_HUMAN Small inducible cytokine A2precursor B3AR_HUMAN Beta-3 adrenergic receptor TSHR_HUMAN Thyrotropinreceptor precursor CB1R_HUMAN Cannabinoid receptor 1 DADR_HUMAN D(1A)dopamine receptor LSHR_HUMAN Lutropin-choriogonadotropic hormonereceptor precursor HH2R_HUMAN Histamine H2 receptor NY1R_HUMANNeuropeptide Y receptor type 1 5H1D_HUMAN 5-hydroxytryptamine 1Dreceptor 5H1B_HUMAN 5-hydroxytryptamine 1B receptor 5H1E_HUMAN5-hydroxytryptamine 1E receptor SSR1_HUMAN Somatostatin receptor type 1SSR2_HUMAN Somatostatin receptor type 2 5H1F_HUMAN 5-hydroxytryptamine1F receptor SSR4_HUMAN Somatostatin receptor type 4 VIPR_HUMANVasoactive intestinal polypeptide receptor 1 precursor CKR1_HUMAN C-Cchemokine receptor type 1 SSR3_HUMAN Somatostatin receptor type 3MC5R_HUMAN Melanocortin-5 receptor 5H7_HUMAN 5-hydroxytryptamine 7receptor CB2R_HUMAN Cannabinoid receptor 2 CRF1_HUMAN Corticotropinreleasing factor receptor 1 precursor SSR5_HUMAN Somatostatin receptortype 5 OPRM_HUMAN Mu-type opioid receptor OPRD_HUMAN Delta-type opioidreceptor MC3R_HUMAN Melanocortin-3 receptor PI2R_HUMAN Prostacyclinreceptor CXC1_HUMAN Chemokine XC receptor 1 ML1A_HUMAN Melatoninreceptor type 1A ML1B_HUMAN Melatonin receptor type 1B 5H6_HUMAN5-hydroxytryptamine 6 receptor ACTR_HUMAN Adrenocorticotropic hormonereceptor MSHR_HUMAN Melanocyte stimulating hormone receptor PTRR_HUMANParathyroid hormone/parathyroid hormone-related Peptide receptorprecursor 5H4_HUMAN 5-hydroxytryptamine 4 receptor CGRR_HUMAN Calcitoningene-related peptide type 1 receptor precursor EDG7_HUMANLysophosphatidic acid receptor Edg-7 HH3R_HUMAN Histamine H3 receptorHtr7 RGD 5-hydroxytryptamine (serotonin) receptor 7 G-protein signaling,coupled to cAMP nucleotide second messenger PE23_MOUSE Prostaglandin E2receptor, EP3 subtype CYA4_MOUSE Adenylate cyclase, type IV P2YC_MOUSEP2Y purinoceptor 12 GALS_HUMAN Galanin receptor type 2 GLP2_HUMANGlucagon-like peptide 2 receptor precursor CAL0_HUMAN Calcitoninprecursor [Contains: Calcitonin; Katacalcin SLIB_HUMAN Somatoliberinprecursor CAL1_HUMAN Calcitonin gene-related peptide I precursorB2AR_HUMAN Beta-2 adrenergic receptor ACM2_HUMAN Muscarinicacetylcholine receptor M2 B3AR_HUMAN Beta-3 adrenergic receptorFMLR_HUMAN fMet-Leu-Phe receptor A1AD_HUMAN Alpha-1D adrenergic receptorAA2A_HUMAN Adenosine A2a receptor V2R_HUMAN Vasopressin V2 receptorPE23_MOUSE Prostaglandin E2 receptor, EP3 subtype MC4R_HUMANMelanocortin-4 receptor GRK5_HUMAN G protein-coupled receptor kinaseGRK5 CRF1_HUMAN Corticotropin releasing factor receptor 1 precursorA1AB_HUMAN Alpha-1B adrenergic receptor PE24_HUMAN Prostaglandin E2receptor, EP4 subtype GLR_HUMAN Glucagon receptor precursor CKR3_HUMANC-C chemokine receptor type 3 CRF2_HUMAN Corticotropin releasing factorreceptor 2 precursor Q8BZV8 P2Y12 platelet ADP receptor homologCYA4_MOUSE Adenylate cyclase, type IV Q99188 ORF OR107W from chromosomeXV P2YC_MOUSE P2Y purinoceptor 12 WAS2_HUMAN Wiskott-Aldrich syndromeprotein family member 2 Adcy2 MGI adenylate cyclase 2 Adcy4 MGIadenylate cyclase 4 P2ry12 MGI With purinergic receptor P2Y, G-proteincoupled 12 Ptger3 MGI prostaglandin E receptor 3 (subtype EP3) Crhr1 RGDI corticotropin releasing hormone 1 CYR1 SGD adenylate cyclase RGS2 SGDGTPase activating protein (GAP) acy-1 IMP - [cgc3038] acy-2 IMP -[cgc3207] C44F1.5 [cgc3038] acy-4 [cgc3207] G-protein signaling,adenylate cyclase activating pathway GBQ_MOUSE Guaninenucleotide-binding protein G(q), alpha subunit Q9D1X2 Thyroidstimulating hormone, receptor TSHR_MOUSE Thyrotropin receptor precursorQ9WUC0 Extra large alpha stimulating guanine-nucleotide Bindingpolypeptide Q9Z0H2 Neuroendocrine-specific golgi protein P55 isoform 2Q9Z0L1 G protein-coupled receptor precursor Q9Z1N8 G-protein XLalphasQ9Z1R7 Guanine nucleotide-binding protein AA2A_MOUSE Adenosine A2areceptor LGR8_MOUSE Relaxin receptor 2 UCN3_MOUSE Urocortin IIIprecursor RAS1_YEAST Ras-like protein 1 GBQ_MOUSE Guaninenucleotide-binding protein G(q), alpha subunit CALR_HUMAN Calcitoninreceptor precursor GPR3_HUMAN Probable G protein-coupled receptor GPR3TSHR_MOUSE Thyrotropin receptor precursor Q14455 Alpha subunit of GsGTPbinding protein (Fragment) AA2A_MOUSE Adenosine A2a receptor GBAF_MOUSEGuanine nucleotide-binding protein G(OLF), Alpha subunit (Fragment)GB10_MOUSE Guanine nucleotide-binding protein, alpha-10 subunitFragment) Q80ZK6 Similar to GNAS (Fragment) Q8BIR3 XLALPHAS proteinhomolog Q8BM77 Similar to G protein coupled receptor AFFECTINGtesticular DESCENT Q8BUB2 GNAS Q8BXD1 Similar to G protein coupledreceptor AFFECTING testicular DESCENT Q8C6E2 Inferred: endothelialdifferentiation Q8CAU3 Adenosine A2a receptor (Fragment) LGR8_MOUSERelaxin receptor 2 UCN3_MOUSE Urocortin III precursor Q9D1X2 Thyroidstimulating hormone, receptor Q9D697 Thyroid stimulating hormone,receptor Q9JJX0 Xlalphas protein (Fragment) Q9QXW5 Nesp Q9QYZ0 Extralarge alpha stimulating guanine-nucleotide binding protein (Fragment)Q9WUC0 Extra large alpha stimulating guanine-nucleotide bindingpolypeptide Q9Z0H2 Neuroendocrine-specific golgi protein P55 isoform 2Q9Z0L1 G protein-coupled receptor precursor Q9Z1N8 G-protein XLalphasQ9Z1R7 Guanine nucleotide-binding protein Adora2a adenosine A2a receptorEdg6 endothelial differentiation, G-protein-coupled receptor 6 Gnalguanine nucleotide binding protein, alpha stimulating, olfactory typeGnaq guanine nucleotide binding protein, alpha q polypeptide Gnas GNAS(guanine nucleotide binding protein, alpha stimulating) complex locusGnas GNAS (guanine nucleotide binding protein, alpha stimulating)complex locus Gpr106 G protein-coupled receptor 106 Ptger4 prostaglandinE receptor 4 (subtype EP4) Tshr thyroid stimulating hormone receptorUcn3 urocortin 3 RAS1 ras homolog adenylate cyclase activation piaAcytosolic regulator of adenylyl cyclase pianissimo G-salpha60AG-salpha60A Pacap38 F Pacap38 DADR_MOUSE D(1A) dopamine receptor O43190Not Available CAL0_HUMAN Calcitonin precursor [Contains: Calcitonin;Katacalcin GBAS_HUMAN Guanine nucleotide-binding protein G(S), alphasubunit CAL1_HUMAN Calcitonin gene-related peptide I precursorB2AR_HUMAN Beta-2 adrenergic receptor B1AR_HUMAN Beta-1 adrenergicreceptor PACA_HUMAN Pituitary adenylate cyclase activating polypeptideprecursor ET1R_HUMAN Endothelin-1 receptor precursor AA2A_HUMANAdenosine A2a receptor AA2B_HUMAN Adenosine A2b receptor V2R_HUMANVasopressin V2 receptor CALR_HUMAN Calcitonin receptor precursorAA3R_HUMAN Adenosine A3 receptor CRF1_HUMAN Corticotropin releasingfactor receptor 1 precursor CAP2_HUMAN Adenylyl cyclase-associatedprotein 2 GLP1_HUMAN Glucagon-like peptide 1 receptor precursorGIPR_HUMAN Gastric inhibitory polypeptide receptor precursor CAP1_HUMANAdenylyl cyclase-associated protein 1 GRFR_HUMAN Growthhormone-releasing hormone receptor precursor GBAF_MOUSE Guaninenucleotide-binding protein G(OLF), alpha sub-unit (Fragment) GB10_MOUSEGuanine nucleotide-binding protein, alpha-10 subunit (Fragment)DADR_MOUSE D(1A) dopamine receptor B2AR_ONCMY Beta-2 adrenergic receptorAdcy1 adenylate cyclase 1 Adcy2 adenylate cyclase 2 Adcy3 adenylatecyclase 3 Adcy4 adenylate cyclase 4 Adcy5 adenylate cyclase 5 Adcy6adenylate cyclase 6 Adcy7 adenylate cyclase 7 Adcy9 adenylate cyclase 9Adcyap1 adenylate cyclase activating polypeptide 1 Drd1a dopaminereceptor D1A Gnal guanine nucleotide binding protein, alpha stimulating,olfactory type RAS2 small GTP-binding protein dopamine receptor,adenylate cyclase activating pathway Q8CH75 Mu opioid receptor variant PQ8VI69 Mu opioid receptor variant BII OPRM_MOUSE Mu-type opioid receptorQ9JIY1 Mu opioid receptor variant F Q9R0D1 Mu opioid receptor variant CQ9R1L9 Mu opioid receptor MOR1E DADR_MOUSE D(1A) dopamine receptorDADR_HUMAN D(1A) dopamine receptor DBDR_HUMAN D(1B) dopamine receptorOPRM_MOUSE Mu-type opioid receptor DADR_MOUSE D(1A) dopamine receptorQ8CAN5 Opioid receptor Q8CGW2 Mu opioid receptor variant MOR-1R Q8CH73Mu opioid receptor variant R Q8CH74 Mu opioid receptor variant Q Q8CH75Mu opioid receptor variant P Q8VI69 Mu opioid receptor variant BIIQ8VI70 Mu opioid receptor variant BI Q8VI71 Mu opioid receptor variant AOPR2_MOUSE Mu-type opioid receptor, isoforms 1G to 1M Q9JIY1 Mu opioidreceptor variant F Q9R0D1 Mu opioid receptor variant C Q9R0D2 Mu opioidreceptor variant 110222 (Fragment) Q9R1L9 Mu opioid receptor MOR1EQ9R1M0 Mu opioid receptor MOR1D Drd1a dopamine receptor D1A NOT Oprd1opioid receptor, delta 1 Oprm opioid receptor, mu Tar1 trace aminereceptor 1 Serotonin receptor, adenylate cyclase activating pathway5-HT7 5-HT7 5-HT7 5-HT7 5-HT7 5-HT7 Htr7 5-hydroxytryptamine (serotonin)receptor 7 G-protein signaling, adenylate cyclase inhibiting pathwayGBI2_MOUSE Guanine nucleotide-binding protein G(i), alpha-2 subunitQ8CH75 Mu opioid receptor variant P Q8VI69 opioid receptor variant BIISSR2_MOUSE Somatostatin receptor type 2 OPRD_MOUSE Delta-type opioidreceptor Q9DC35 Endothelial differentiation sphingolipidG-protein-coupled receptor 1 OPRM_MOUSE Mu-type opioid receptor Q9JIY1Mu opioid receptor variant F Q9R0D1 Mu opioid receptor variant C Q9R1L9Mu opioid receptor MOR1E Q9Z0U9 LYSOPHOSPHOLIPID receptor B3 EDG1_MOUSEProbable G protein-coupled receptor Edg-1 CORT_HUMAN Cortistatinprecursor [Contains: Cortistatin-29; Cortistatin-17] EDG1_MOUSE ProbableG protein-coupled receptor Edg-1 GBI2_MOUSE Guanine nucleotide-bindingprotein G(i), alpha-2 subunit NY1R_HUMAN Neuropeptide Y receptor type 1SSR2_MOUSE Somatostatin receptor type 2 OPRD_MOUSE Delta-type opioidreceptor OPRK_HUMAN Kappa-type opioid receptor OPRX_HUMAN Nociceptinreceptor OPRM_MOUSE Mu-type opioid receptor NY2R_HUMAN Neuropeptide Yreceptor type 2 RGS1_HUMAN Regulator of G-protein signaling 1 Q8BLP9Delta-type opioid receptor Q8BP20 Endothelial differentiation Q8C4A3Endothelial differentiation sphingolipid G-protein-coupled receptor 1Q8CAN5 Opioid receptor Q8CGW2 Mu opioid receptor variant MOR-1R Q8CH73Mu opioid receptor variant R Q8CH74 Mu opioid receptor variant Q Q8CH75Mu opioid receptor variant P Q8JZT4 Similar to guanine nucleotidebinding protein, alpha inhibiting 2 Q8VI69 Mu opioid receptor variantBII Q8VI70 Mu opioid receptor variant BI Q8VI71 Mu opioid receptorvariant A OPR2_MOUSE Mu-type opioid receptor, isoforms 1G to 1M Q922Y6Hypothetical protein (Fragment) MCR1_HUMAN Melanin-concentrating hormonereceptor 1 Q9DC35 Endothelial differentiation sphingolipidG-protein-coupled receptor 1 Q9JIY1 Mu opioid receptor variant F Q9R0D1Mu opioid receptor variant C Q9R0D2 Mu opioid receptor variant 110222(Fragment) Q9R1L9 Mu opioid receptor MOR1E Q9R1M0 Mu opioid receptorMOR1D Q9R235 Lysophospholipid receptor B1 Q9Z0U9 LYSOPHOSPHOLIPIDreceptor B3 Edg1 endothelial differentiation sphingolipidG-protein-coupled receptor 1 Edg3 endothelial differentiation,sphingolipid G-protein-coupled receptor, Gnai2 guanine nucleotidebinding protein, alpha inhibiting 2 Npb neuropeptide B Oprd1 opioidreceptor, delta 1 Oprm opioid receptor, mu Sstr2 somatostatin receptor 2Oprm1 “Opioid receptor, mu 1” dopamine receptor, adenylate cyclaseinhibiting pathway D2DR_HUMAN D(2) dopamine receptor muscarinic acetylcholine receptor, adenylate cyclase inhibiting pathway ACM2_HUMANMuscarinic acetylcholine receptor M2 ACM5_HUMAN Muscarinic acetylcholinereceptor M5 Negative regulation of adenylate cyclase activity MGR8_HUMANMetabotropic glutamate receptor 8 precursor GALT_HUMAN Galanin receptortype 3 GBR2_HUMAN Gamma-aminobutyric acid type B receptor, subunit 2precursor GBI2_HUMAN Guanine nucleotide-binding protein G(i), alpha-2subunit GBAK_HUMAN Guanine nucleotide-binding protein G(k), alphasubunit A2AA_HUMAN Alpha-2A adrenergic receptor ETBR_HUMAN Endothelin Breceptor precursor CKR2_HUMAN C-C chemokine receptor type 2 GALR_HUMANGalanin receptor type 1 MGR2_HUMAN Metabotropic glutamate receptor 2precursor MGR7_HUMAN Metabotropic glutamate receptor 7 precursorMGR3_HUMAN Metabotropic glutamate receptor 3 precursor MGR4_HUMANMetabotropic glutamate receptor 4 precursor Q9NPE5 Not AvailableGBR1_HUMAN Gamma-aminobutyric acid type B receptor, subunit 1 precursorpositive regulation of adenylate cyclase activity dagA cytosolicregulator of adenylyl cyclase serotonin receptor, adenylate cyclaseinhibiting pathway 5-HT1A 5-HT1A 5-HT1B 5-HT1B 5HTA_DROME5-hydroxytryptamine receptor 2A 5HTB_DROME 5-hydroxytryptamine receptor2B Q9V8Q3 CG15113-PA Q9V8Q9 CG16720 protein G-protein signaling, coupledto cGMP nucleotide second messenger TBL3_HUMAN WD-repeat protein SAZD4933400B15Rik RIKEN cDNA 4933400B15 gene Gnat1 guanine nucleotidebinding protein, alpha transducing 1 Gnat2 guanine nucleotide bindingprotein, alpha transducing 2 Tbl3 transducin (beta)-like 3 Nos2 nitricoxide synthase 2 G-protein signaling, coupled to IP3 second messenger(phospholipase C activating) GB15_MOUSE Guanine nucleotide-bindingprotein, alpha-15 subunit Q9ERT2 Thyrotropin-releasing hormone receptor2 GBGD_MOUSE Guanine nucleotide-binding protein G(I)/G(S)/G(O) gamma-13subunit O43190 Not Available O76067 Not Available ETBR_HUMAN EndothelinB receptor precursor IL8B_HUMAN High affinity interleukin-8 receptor BNK1R_HUMAN Substance-P receptor NMBR_HUMAN Neuromedin-B receptorAG2R_HUMAN Type-1 angiotensin II receptor PE23_MOUSE Prostaglandin E2receptor, EP3 subtype OXYR_HUMAN Oxytocin receptor GB15_MOUSE Guaninenucleotide-binding protein, alpha-15 subunit CCKR_HUMAN Cholecystokinintype A receptor GASR_HUMAN Gastrin/cholecystokinin type B receptorHH1R_HUMAN Histamine H1 receptor P2Y2_HUMAN P2Y purinoceptor 25H2B_HUMAN 5-hydroxytryptamine 2B receptor MC3R_HUMAN Melanocortin-3receptor P2YR_HUMAN P2Y purinoceptor 1 P2Y4_HUMAN P2Y purinoceptor 4P2Y6_HUMAN P2Y purinoceptor 6 L4R1_HUMAN Leukotriene B4 receptor 1Q61621 G-protein beta subunit (Fragment) Q9ERT1 Thyrotropin-releasinghormone receptor 2 (Fragment) Q9ERT2 Thyrotropin-releasing hormonereceptor 2 GBGD_MOUSE Guanine nucleotide-binding protein G(I)/G(S)/G(O)gamma-13 subunit Q9NYK7 CCK-B/gastrin receptor Gna15 guanine nucleotidebinding protein, alpha 15 Gnb1 guanine nucleotide binding protein, beta1 Gng13 guanine nucleotide binding protein 13, gamma Ptger3prostaglandin E receptor 3 (subtype EP3) Trhr2 thyrotropin releasinghormone receptor 2 Agtr1a angiotensin receptor 1a cytosolic calcium ionconcentration elevation norpA norpA Q99L49 Similar to transient receptorprotein 2 PE23_MOUSE Prostaglandin E2 receptor, EP3 subtype TRP6_MOUSEShort transient receptor potential channel 6 JPH2_MOUSE Junctophilin 2SY28_MOUSE Small inducible cytokine A28 precursor CKRA_MOUSE C-Cchemokine receptor type 10 TRP2_MOUSE Short transient receptor potentialchannel 2 O43431 Not Available GALS_HUMAN Galanin receptor type 2SZ13_HUMAN Small inducible cytokine B13 precursor O95977 PutativeG-protein coupled receptor, EDG6 precursor CAL0_HUMAN Calcitoninprecursor [Contains: Calcitonin; Katacalcin CAL1_HUMAN Calcitoningene-related peptide I precursor DADR_HUMAN D(1A) dopamine receptorC5AR_HUMAN C5a anaphylatoxin chemotactic receptor ET1R_HUMANEndothelin-1 receptor precursor BRB2_HUMAN B2 bradykinin receptorAG2R_HUMAN Type-1 angiotensin II receptor PE23_MOUSE Prostaglandin E2receptor, EP3 subtype CCR4_HUMAN C—X—C chemokine receptor type 4CCKR_HUMAN Cholecystokinin type A receptor GASR_HUMANGastrin/cholecystokinin type B receptor CKR1_HUMAN C-C chemokinereceptor type 1 CKR7_HUMAN C-C chemokine receptor type 7 precursorV1AR_HUMAN Vasopressin V1a receptor CKR2_HUMAN C-C chemokine receptortype 2 CXC1_HUMAN Chemokine XC receptor 1 BRB1_HUMAN B1 bradykininreceptor V1BR_HUMAN Vasopressin V1b receptor CCR3_HUMAN C—X—C chemokinereceptor type 3 P2Y4_HUMAN P2Y purinoceptor 4 CKR3_HUMAN C-C chemokinereceptor type 3 CKR4_HUMAN C-C chemokine receptor type 4 CKR5_HUMAN C-Cchemokine receptor type 5 (CCR5) CKR6_HUMAN C-C chemokine receptor type6 CKR8_HUMAN C-C chemokine receptor type 8 CKR9_HUMAN C-C chemokinereceptor type 9 PAR2_HUMAN Proteinase activated receptor 2 precursorC3AR_HUMAN C3a anaphylatoxin chemotactic receptor Q61057 Trp-relatedprotein 2 (Fragment) TRP6_MOUSE Short transient receptor potentialchannel 6 Q8BRU2 Transient receptor protein 2 Q8CDC6 Transient receptorprotein 2 Q8CEM7 Transient receptor protein 2 (Fragment) EDG2_HUMANLysophosphatidic acid receptor Edg-2 EDG3_HUMAN Sphingosine 1-phosphatereceptor Edg-3 MCR1_HUMAN Melanin-concentrating hormone receptor 1Q99L49 Similar to transient receptor protein 2 JPH2_MOUSE Junctophilin 2SY28_MOUSE Small inducible cytokine A28 precursor CKRA_MOUSE C-Cchemokine receptor type 10 Q9NYK7 CCK-B/gastrin receptor TRP2_MOUSEShort transient receptor potential channel 2 EDG7_HUMAN Lysophosphatidicacid receptor Edg-7 CLT1_HUMAN Cysteinyl leukotriene receptor 1 Ccl28chemokine (C-C motif) ligand 28 Edg3 endothelial differentiation,sphingolipid G-protein-coupled receptor, 3 Gpr2 G protein-coupledreceptor 2 Jph2 junctophilin 2 Ptger3 prostaglandin E receptor 3(subtype EP3) Trpc2 transient receptor potential cation channel,subfamily C, member 2 Trpc6 transient receptor potential cation channel,subfamily C, member 6 Itpr3 inositol 1, 4, 5-triphosphate receptor 3″Trrp6 “transient receptor potential cation channel, subfamily C, member6” itr-1 Not Available Metabotropic glutamate receptor, phospholipase Cactivating pathway O96003 SYN47 protein MGR5_HUMAN Metabotropicglutamate receptor 5 precursor Grm5 “glutamate receptor, metabotropic 5”Muscarinic acetyl choline receptor, phospholipase C activating pathwayACM2_HUMAN Muscarinic acetylcholine receptor M2 ACM1_HUMAN Muscarinicacetylcholine receptor M1 (herein also designated Muscarinic M1)GB15_HUMAN Guanine nucleotide-binding protein, alpha-15 subunit Chrm3cholinergic receptor, muscarinic 3, cardiac phospholipase C activationGalpha49B Galpha49B Gbeta76C Gbeta76C O43431 Not Available O95977Putative G-protein coupled receptor, EDG6 precursor CAL0_HUMANCalcitonin precursor [Contains: Calcitonin; Katacalcin CAL1_HUMANCalcitonin gene-related peptide I precursor C5AR_HUMAN C5a anaphylatoxinchemotactic receptor GBQ_DROME Guanine nucleotide-binding protein G(q),alpha subunit ET1R_HUMAN Endothelin-1 receptor precursor GBB2_DROMEGuanine nucleotide-binding protein beta subunit 2 GB15_HUMAN Guaninenucleotide-binding protein, alpha-15 subunit CALR_HUMAN Calcitoninreceptor precursor GASR_HUMAN Gastrin/cholecystokinin type B receptorV1AR_HUMAN Vasopressin V1a receptor V1BR_HUMAN Vasopressin V1b receptorGBQ_HUMAN Guanine nucleotide-binding protein G(q), alpha subunitPIB2_HUMAN 1-phosphatidylinositol-4,5-bisphosphate phospho diesterasebeta 2 EDG2_HUMAN Lysophosphatidic acid receptor Edg-2 Q9I7C8 G proteinalpha 49B Q9NYK7 CCK-B/gastrin receptor Q9TXA4 Signal-transducing Gprotein alpha Q subunit Q9VW29 GBETA76C protein Adra1a “adrenergicreceptor, alpha 1a” Adcyap1r1 adenylate cyclase activating polypeptide 1receptor 1 protein kinase C activation PF14_0681 diacylglycerol kinase,putative Pfalciparum GBLP_HUMAN Guanine nucleotide-binding protein betasubunit-like protein 12.3 PIC1_MOUSE PRKCA-binding protein GBLP_HUMANGuanine nucleotide-binding protein beta subunit-like protein 12.3PIC1_RAT PRKCA-binding protein KPCN_HUMAN Protein kinase C, nu typeACM1_HUMAN Muscarinic acetylcholine receptor M1 NEUM_HUMAN NeuromodulinCAP7_HUMAN Azurocidin precursor ET2_HUMAN Endothelin-2 precursorGBLP_HUMAN Guanine nucleotide-binding protein beta subunit-like protein12.3 143F_HUMAN 14-3-3 protein eta PIC1_MOUSE PRKCA-binding proteinQ80VC8 Similar to protein that interacts with C kinase 1 Q8C1W2 Proteinthat interacts with C kinase 1 PIC1_RAT PRKCA-binding protein PIC1_HUMANPRKCA-binding protein C130010K08Rik RIKEN cDNA C130010K08 gene Cerkceramide kinase Gnb2-rs1 guanine nucleotide binding protein, beta 2,related sequence 1 F13G24.120 diacylglycerol kinase 1 (DGK1) F17I23.320diacylglycerol kinase family F18E5.160 diacylglycerol kinase familyF26K10.10 diacylglycerol (DAG) kinase accessory domain protein F5H14.13diacylglycerol kinase, putative K19M13.8 diacylglycerol kinase familyMBK5.25 diacylglycerol kinase, putative MRI1.5 diacylglycerol kinase,putative MSF3.11 diacylglycerol kinase, putative F13G24.120diacylglycerol kinase 1 (DGK1) F26K10.10 diacylglycerol (DAG) kinaseaccessory domain protein T3F17.26 diacylglycerol kinase family serotoninreceptor, phospholipase C activating pathway 5-HT1A 5-HT1A 5-HT1A 5-HT1A5-HT1B 5-HT1B 5-HT1B 5-HT1B 5HTA_DROME 5-hydroxytryptamine receptor 2A5HTB_DROME 5-hydroxytryptamine receptor 2B Q9V8Q3 CG15113-PA Q9V8Q9CG16720 protein Htr2b 5-hydroxytryptamine (serotonin) receptor 2B Genesymbol refers to the symbol used in the Gene Ontology Blast serveravailable 25 May 2005 at http://godatabase.org/cgi-bin/go.cgi?view=blast&session_id=87201075891145.

In an even further embodiment of the invention the GPCR is coupled to aG-protein, such as G_(Q), that activates phospholipase C. Examples ofsuch GPCRs are given in table 3.

Other receptors than GPCR may be used with the present invention, forexample the cell surface molecule may be a receptor selected from thegroup consisting of receptors belonging to the family of protein kinasecoupled receptors and receptors belonging to the family of receptorkinases.

The family of Protein kinase coupled receptors for example includesreceptors for cytokines, interferons and HGF. These receptors do nothave intrinsic kinase activity, but are associated with a kinase.

Activation of preferred protein kinase coupled receptors results inactivation of AP-1, i.e. in increased transcription from genescomprising one or more AP-1 sites in their regulatory sequences. This isin particular true for receptors activated by a cytokine.

Receptor kinases are receptors having an intrinsic kinase activity.Frequently said activity may be modulated by association of a ligand.The family for example includes receptors for Insulin, NGF, PDGF, FGF,EGF and GH.

Activation of preferred receptor kinases results in activation of SRE,i.e. in an increase in transcription from genes comprising one or moreSRE in their regulatory sequences. This is in particular true forreceptor kinases activated by growth hormones.

The receptor may also be an orphan receptor, i.e. a receptor for whichno ligand has yet been identified. The methods of the present inventionmay also be useful for identifying ligands of orphan receptors.

The cell surface molecule may in one embodiment of the invention be achannel which is accessible from the extracellular surface, such as atransmembrane channel. Examples of channels are ion-channels, such asCa²⁺ channels.

Cellular Response

The invention relates to methods of identifying compounds modulating,such as activating or inhibiting, a cellular response linked to areporter system. The reporter system may be any of the reporter systemsdescribed herein below. The methods disclosed by the present inventionmay be used to identify compounds modifying any cellular response, whichis or may be linked to a reporter system generating a detectable output.The person skilled in the art will appreciate that the specific methodsdisclosed herein may be adapted to any such cellular response. Below,non-limiting examples of cellular responses are described.

In a particularly preferred embodiment of the invention, the cellularresponse is mediated through a cell surface molecule, for example thecellular response may be activation of a receptor. Hence, the cellularresponse may for example be modulation of a signal transduction pathwaywithin a cell, such as modulation of a signal transduction pathwaymediated by a cell surface molecule. By “activation of a receptor” ismeant that the receptor is influenced in a manner that it activatesdownstream signalling events. Accordingly, the methods according to thepresent invention may be employed to identify agonists or antagonist ofa receptor.

Examples includes:

-   -   Upregulation or downregulation of the level of a member of the        pathway    -   Relocalisation of a member of the pathway    -   Complex formation between members of the pathway or between        members of the pathway with other cellular compounds    -   Enhanced or reduced transcription from genes regulated by the        pathway    -   Modification by for example phosphorylation of a member of the        pathway    -   Activation or inhibition of an enzyme of the pathway    -   Degradation of a cellular compounds due to upregulation or        downregulation of the pathway    -   Altered secretion of a compound    -   Change in ion-flux    -   Morphological changes    -   Change in viability

In a preferred embodiment the signal transduction pathway is a pathwaymodulated by any of the receptors described in the section herein above.Hence, the cellular response may for example be any of the following:

-   -   Activation of adenylate cyclase; i.e. increase in adenylate        cyclase activity    -   Increased levels of cAMP    -   Increased transcription of genes regulated by a CRE    -   Inhibition of adenylate cyclase; i.e. decrease in adenylate        cyclase activity    -   Decreased levels of cAMP    -   Decreased transcription of genes regulated by a CRE    -   Increased activity of phospholipase C    -   Increased level of inositol 1,4,5-trisphosphate    -   Increased activity of Protein kinase C(PKC)    -   Phosphorylation of proteins, which are phosphorylated by protein        kinase C

The cellular response may in one embodiment be modulation oftranscriptional activity, such as activation or reduction oftranscription of one or more genes. In particular, activation orreduction of transcription of genes regulated by a response element.Said response element could for example be selected from the groupconsisting of CRE, SRE, TRE and AP-1.

Hence, the cellular response may also be an increased or decreased levelof a particular mRNA within a cell.

By the term “regulated by a response element” is meant thattranscription is modulated by said response element, however otherelements may also modulate transcription of said gene. By the term“activation of response element” is meant increased transcription ofgenes regulated by said response element and/or operably associatedtherewith.

In another embodiment of the invention the cellular response is:

-   -   change in the intracellular level of a compound; or    -   change in the level of a compound within a specific cellular        compartment, for example within the cytoplasm, in the golgi, in        the endoplasmatic reticulum, in lysosomes, in endosomes or in        the nucleus

The compound may be any compound, preferably a naturally occurringcompound. Frequently, the compound is a compound endogenous to the cell.The compound may thus for example be a salt, an ion, a nucleotide or aderivative thereof, a peptide, a saccharide, a lipid or abiomacromolecule. Biomacromolecules includes for example RNA such asmRNA, polypeptides and proteins. An example of an ion is Ca²⁺ and anexample of a nucleotide derivative is cAMP.

In yet another embodiment of the invention the cellular response isrelocalisation of a compound. Relocalisation may for example be

-   -   concentration of a compound otherwise dispersed in one or more        specific locations    -   relocalisation from one cellular compartment to another, for        example relocalisation from the cellular membrane to the        cytoplasma.    -   relocalisation from one location within a compartment to another        location within the same compartment    -   internalisation of an extracellular compound

The compound may be any compound, such as any of the compounds mentionedin the section above. In one preferred embodiment the compound, which isrelocalised is a biomacromolecule, such as RNA, polypeptides orproteins. For example, the compound may be a cell surface receptor(receptor). The cellular response may thus be internalisation of saidreceptor or relocalisation of said receptor from the cellular membraneto the cytoplasma.

In one embodiment of the invention the cellular response is change inthe activity of a compound, such as an increase or a decrease in theactivity of a compound. Said compound may for example be an enzyme.

In another embodiment of the invention the cellular response is changein phosphorylation of a compound.

In another embodiment of the invention the cellular response isformation or disruption of a complex between compounds.

In another embodiment of the invention the cellular response is changein the concentration of a compound.

The cellular response may also be altered secretion of a compound, suchas increased or decreased secretion of a compound. Said compound couldfor example be a biomacromolecule, such as a protein, a polypeptide, apeptide, a hormone, a cytokine, or the like.

In another embodiment of the invention the cellular response is changein pH in an intracellular compartment, for example in the cytoplasm.

In yet another embodiment the cellular response is a change in amembrane potential, for example a change in membrane potential over thecell membrane or over the mitochondria membrane.

In an even further embodiment of the invention the cellular response ischange in morphology, such as change in size or shape. The cellularresponse may also be change in viability (e.g. apoptosis or necrosis)under specific conditions.

The methods according to the invention may also include identificationof compounds modulating more than one cellular response, such as 2, forexample 3, such as 4, for example 5, such as more than 5 differentcellular responses. Said cellular responses may be any of the responsesdiscussed above.

Reporter System

The reporter system to be used with the present invention should beselected according to the particular cellular response. The reportersystem should be capable of generating a detectable output.

In some embodiments of the invention the reporter system may beidentical to the cellular response. This is in particular true when thecellular response may be detected without the aid of an additionalreporter system, for example when the cellular response is anincrease/decrease in the level of a compound, relocalisation of acompound, change in membrane potential, change in pH, change inmorphology or the like.

Hence, the reporter system may be a system endogenous to said cells. Forexample, the reporter system may comprise the endogenous systemregulating the intra-cellular level of an endogenous compound. By way ofexample, the reporter system may be the endogenous system of a cellregulating the intracellular Ca²⁺ level.

In another example, the reporter system comprises the intracellularlocalisation of an endogenous compound.

However, the reporter system may also be heterologous to the cell, i.e.a reporter system which has been inserted into the cell for example byrecombinant techniques.

In embodiments of the invention, wherein the cellular response ismodulation of transcription from gene(s) regulated by a responseelement, it is preferred that the report system comprises a nucleic acidcomprising a nucleotide sequence encoding a detectable polypeptideoperably linked to a response element, the activity of which ismodulated by the cellular response.

In embodiments of the invention, wherein the cellular response ismodulation of a signal transduction pathway, the reporter system maycomprise a nucleic acid comprising a nucleotide sequence encoding adetectable polypeptide operably linked to a response element, theactivity of which is modulated by said signal transduction pathway.

For example, if the cellular response is modulation of a signaltransduction pathway influencing the activity of CRE and/or SRE, thenthe reporter system may comprise a nucleic acid comprising a nucleotidesequence encoding a detectable polypeptide operably linked to a responseelement selected from the group consisting of cAMP response element(CRE) and serum response element (SRE). Examples of such signaltransduction pathways include the signal transduction pathways modulatedby GPCR of the rhodopsin family or secretin family and by protein kinasereceptors and receptors belonging to the family of receptor kinases.

By way of example: 1) If the cellular response is activation of a signaltransduction pathway activated by a GPCR coupled to a G_(S) (see hereinabove) that stimulates adenylate cyclase, then the reporter system maybe a nucleic acid comprising a nucleotide sequence encoding a detectablepolypeptide operably linked to CRE. Activation of said GPCR may then bedetected by detection of increased levels of said detectablepolypeptide. 2) If the cellular response is activation of signaltransduction pathway activated by a GPCR coupled to a G_(I) (see hereinabove) that inhibits adenylate cyclase, then the reporter system may bea nucleic acid comprising a nucleotide sequence encoding a detectablepolypeptide operably linked to CRE. Activation of said GPCR may then bedetected by detection of decreased levels of said detectablepolypeptide.

Similarly, if the cellular response is modulation of a signaltransduction pathway that influences the activity of TRE, then thereporter system may comprise a nucleic acid comprising a nucleotidesequence encoding a detectable polypeptide operably linked to TPAresponse element (TRE). Examples are GPCRs that are linked to activationof Protein Kinase C such as Gq coupled receptors (see herein above).

Similarly, if the cellular response is modulation of a signaltransduction pathway that influences the activity of SRE, then thereporter system may comprise a nucleic acid comprising a nucleotidesequence encoding a detectable polypeptide operably linked to SRE.Examples of such signal transduction pathways include the signaltransduction pathways modulated by growth hormones or cytokines throughprotein kinase receptors and receptors belonging to the family ofreceptor kinases.

Similarly, if the cellular response is modulation of a signaltransduction pathway that influences the activity of AP-1, then thereporter system may comprise a nucleic acid comprising a nucleotidesequence encoding a detectable polypeptide operably linked to AP-1.Examples of such signal transduction pathways include the signaltransduction pathways modulated by cytokines or growth factors cytokinesthrough protein kinase receptors and receptors belonging to the familyof receptor kinases

The detectable polypeptide may be any detectable polypeptide, howeverpreferably the detectable polypeptide is selected from the groupconsisting of fluorescent proteins and enzymes.

Fluorescent proteins may for example be green fluorescent protein (GFP)and fluorescent mutants thereof, such as yellow fluorescent protein(YFP) or cyan fluorescent protein (CFP). The fluorescent protein canalso be a protein complex, e.g. a di- or tetramer of a fluorescentprotein, such as dsRed. Enzymes may for'example be selected from thegroup consisting of luciferase, CAT, galactosidase, alkaline phosphataseand beta-lactamase.

In one embodiment of the invention the reporter system may comprise abioluminescent moiety. For example, if the cellular response isrelocalisation of a compound, then the reporter system may for examplebe said compound linked to a luminiscent moiety, such as a fluorescentmoeity. Hence, for example if the cellular response is relocalisation ofa polypeptide the reporter system may be a chimeric protein made up ofsaid polypeptide and a fluorescent protein, such as GFP, YFP or CFP. Inone preferred embodiment said polypeptide may be receptor.

In one embodiment of the invention the reporter system may detectcomplex formation between two cellular proteins. This may for example beachieved by linking a bioluminescent moiety, such as luciferase, to theone protein and a fluorescent moiety, such as a fluorescent protein, tothe other protein. Direct interaction between the proteins can afterexpression of the two chimeric proteins be detected through occurrenceof BRET (Bioluminescence Resonance Energy Transfer). If the two proteinsare linked to fluorescent moieties it is possible to detect the complexformation through the occurrence of FRET (Fluorescence Resonance EnergyTransfer). Complex formation may also be detecting using scintillationproximity assays.

Hence, if the cellular response is relocalisation of a cell surfacemolecule, then the reporter system may comprise a fluorescent moietycovalently coupled to said cell surface molecule.

In some embodiments of the invention the cellular response is modulationof a signal transduction pathway involving activation of phospholipaseC. Phospholipase C may for example be activated by GPCRs coupled toG_(Q) (see herein above). Activation of phospholipase C in general leadsto increase in the intracellular level of Ca²⁺ and thus in suchembodiments the reporter system may be the intracellular Ca²⁺ level.This reporter system may thus be endogenous to the cell.

Detectable Output

The detectable output may be any output, which is detectable directly orindirectly. For example the detectable output may be the concentrationof a compound within a cell, localisation of a compound within a cell,luminiscense, activity of an enzyme or the like:

In preferred embodiments of the invention the detectable output isluminiscense, such as fluorescence, bioluminescence, FRET or BRET.Bioluminiscence may be detected by any conventional methods, for examplewith the aid of a Plate reader. BRET may be performed as describedherein above. In one embodiment, BRET2 technology is used which is basedon energy transfer between a bioluminescent donor (a Renilla luciferase(Rluc) fusion protein) and a fluorescent acceptor (a Green FluorescentProtein (GFP2) fusion protein). In presence of its substrate DeepBlueC™(a coelenterazine derivative), Rluc emits blue light (˜395 nm). Aprotein-protein interaction between Rluc and GFP2 fusion proteins allowsenergy transfer to GFP2 which reemits green light (510 nm). Expressionof Rluc alone, in the presence of the substrate DeepBlueC™, gives anemission spectrum with a peak at ˜395 nm (solid line). With theRluc:GFP2 fusion construct, there is efficient energy transfer betweenRluc and GFP2 and the 510 nm signal represents a major peak (dashedline). The BRET2 signal is expressed as the 515 nm to 410 nm ratio,since filters centered at those wavelengths are used for detection. FRETtechnology is based on the distance-dependent energy transfer betweentwo fluorescence groups that are each coupled to a protein.

Alternatively, the detectable output may preferably be linked (directlyor indirectly) to a bioluminiscent signal.

However, the detectable output could also be radioactivity, a colouredcompound or a colour signal, a heavy metal, an electrical potential, aredox potential, a temperature or the detectable output may be linked toa radioactive signal, a coloured compound or a colour signal or a heavymetal or an electrical potential, or a redox potential or a temperature.Said radioactive signal could for example be ³⁵S, ³²P, ³H. The colouredcompound could for example be the product of any of the enzymaticreaction described herein elsewhere. The heavy metal could for examplebe gold.

In embodiments of the invention, wherein the cellular response is changein the intracellular level of a compound or change in the level of acompound within a specific cellular compartment, then the detectableoutput may be said level of said compound. Depending on the nature ofthe compound, said level may be detected directly or indirectly.

If the compound for example is a fluorescent compound, the level of saidcompound may be determined by determining the fluorescence properties.This may be done by any suitable means, for example by the aid of afluorescence microscope, a FACS (Fluorescence Activated Cell Sorter), aFABS (Fluorescence Activated Bead Sorter), fluorescence plate-reader ora fluorescence spectrometer,

If the compound for example is an enzyme then the level of said compoundmay be determined by determining the activity of said enzyme. By way ofexample, if the enzyme catalyses a reaction leading to a product, whichis directly detectable, for example by colorimetric or chemiluminescentdetection techniques, the activity of said enzyme may be detected bydetecting said compound. For example, if the enzyme is luciferase, theactivity of said enzyme may be detected by detecting emmision of lightupon oxidation of the added substrate, luciferin.

Several other enzymes such as CAT, β-galactosidase, alkalinephosphatase, horseradish peroxidase and beta-lactamase are, whenprovided with suitable substrates, capable of catalysing reactionsleading to coloured or chemiluminescent products, which may be detectedusing any colorimetric or chemiluminescent detection technique.

If the compound for example is Ca²⁺, then the intracellularconcentration of said ion can be measured by using any suitable method,for example by inserting into the cells Ca²⁺ binding fluorescentcompounds like Fura-2, Fluo-3 or Fluo-4 (Molecular Probes), which changefluorescent properties according to a changed Ca²⁺ concentration.Non-limiting examples of methods of determining cytosolic free Ca²⁺ aregiven in examples 13 and 13a herein below. Other ion concentrations canbe monitored using suitable fluorescent compounds, which for example areavailable from Molecular Probes Inc.

If the compound for example is a protein, then it may for example bedetected using a first specific binding partner. Said first specificbinding partner could be a second protein capable of specificallyinteracting with said protein, such as a specific anti-body or saidfirst specific binding partner could be an aptamer. Said first specificbinding partner could be conjugated to a directly detectable compound,such as a fluorescent compound, a radioactive compound or a heavy metalor to an indirectly detectable compound, such as an enzyme, which forexample could be any of the enzymes mentioned herein above. It is alsopossible that the first specific binding partner may be detected with asecond specific binding partner, capable of interacting specificallywith the first specific binding partner. Said second specific bindingpartner may be conjugated to a directly or indirectly detectablecompound similarly to the first specific binding partner. Additionalspecific binding partners may be used.

In embodiments of the invention wherein the cellular response isrelocalisation of a compound the detectable output could be a detectablelabel conjugated to said compound. In particular, the compound may beconjugated to a directly detectable label, such as a fluorescent labelor a heavy metal. Thus the localisation of the compound may be directlydetected, for example using a fluorescence microscope, Fluorescentplate-reader, fluorescence spectrometer, a FACS or a FABS instrument Inone preferred embodiment the compound is a fusion protein comprising aprotein of interest and a fluorescent protein, such as GFP. The compoundmay thus be a fluorescent probe. Thus the detectable output may belocalisation of a fluorescent signal. Alternatively, the compound is afusion protein comprising the protein of interest and a tag. Said tagcould be a tag specifically interacting with a specific binding partner,for example the tag could be an HA-tag or a flash domain. Alternatively,localisation of a compound may be determined with the aid of a specificbinding partner as outlined above. Intracellular localisation may alsobe detected using methods capable of detecting distance between twocompounds, for example BRET or FRET.

In embodiments of the invention wherein the cellular response is changeof activity of a compound, the detectable output may be a product ofsaid activity. I.e. when said compound is an enzyme the detectableoutput could be a product of a reaction catalysed by said enzyme. Saidproduct could thus be a coloured product or a chemiluminiscent productas discussed herein above.

In embodiments of the invention wherein the cellular response isenhanced or reduced transcription from one or more genes, then thecellular response could be mRNA transcribed from said gene, a proteinencoded by said gene or in case the protein is an enzyme, the detectableoutput could be a product of a reaction catalysed by said enzyme. Theenzyme and the products could be any of the enzymes or productsdiscussed herein above.

mRNA may be detected by any useful means, for example with the aid of aprobe capable of hybridising specifically with said mRNA. Said probecould be labelled with a directly detectable label, for example aradioactive compound, a fluorescent compound or a heavy metal or anindirectly detectable label such as an enzyme or a specific bindingpartner.

Said protein may be detected with the aid of specific binding partnersas outlined herein above. However, in a preferred embodiment the proteinis a fluorescent protein and may thus be detected directly. Hence, thedetectable output could be bioluminescence, such as fluorescence.

In embodiments of the invention wherein the cellular response ismodification by for example phosphorylation of a compound this can bedetected through binding of a antibody that specifically bind thephosphorylated protein said antibody can then be quantified by specificfluorescence labelling.

In embodiments of the invention wherein the cellular response is changein pH in an intracellular compartment, the detectable output will ingeneral be said pH. The pH may be determined using any suitable method,for example using a pH indicator or a pH-meter. For example the pH maybe determined using a fluorescent indicator for intracellular pH.Suitable compounds are compounds with a fluorescent excitation profilewhich is pH-dependent, such as BCECF (available from Molecular Probes).In embodiments of the invention wherein the cellular response is achange in a membrane potential, the detectable output will in general besaid membrane potential. The membrane potential may be determined usingany suitable method such as applying a fluorescent molecule to cellsthat distribute over the membrane dependent upon the membrane potential.Examples of such compounds are DiBAC, various ANEP dyes, JC-1 and JC-9(Molecular Probes). For example, JC-1 and JC-9 are cationic dyes thatexhibit potential-dependent accumulation in mitochondria leading to ashift in fluorescence emmision from green to red. Thus mitochondrialdepolarization may for example be determined by decrease in red/greenfluorescence intensity ratio (see also product information fromMolecular Probes). ANEP dyes are in particularly useful for detection ofchanges in membrane potential. The fluorescence can be read for instanceby a fluorescence microscope, a fluorescence plate-reader, a FACS, or aFABS instrument.

In embodiment of the invention wherein the cellular response is changein morphology, the detectable output will in general be the morphologyof the cell. The morphology may be observed using any suitable methodfor example by the aid of a microscope, using a FACS or FABS,

Depending on the detectable output, it will frequently be an advantageto fix cells prior to detecting said detectable output. However, in someembodiments of the invention it is preferred that the cells are notfixed. Cells may be fixed according to any useful protocol (see alsodefinitions herein above).

Selection

The methods according to the invention involves screening resin beadsfor beads comprising cells meeting at least one predetermined selectioncriterion, wherein said selection criterion is linked directly orindirectly to said detectable output. Hence, the selection criterionwill be dependent on the detectable output.

For example the predetermined selection criteria may be a quantitativecriterium, such as a quantitative level of bioluminiscence above orbelow a specific threshold value.

In embodiments of the invention, wherein the detectable output isfluorescence or the detectable output may be linked to a fluorescentsignal, then the predetermined selection criterion could be anyfluorescence property. For example, the selection criterion could beintensity of said fluorescence above or below a predetermined thresholdvalue or emission of light of a specific wavelength or absorption oflight of a specific wavelength or intensity of emitted light of aspecific wavelength above or below a predetermined threshold value. Theselection criterion could also be based on Fluorescence lifetime and/orfluorescence polarization The selection criterion could also be aspecific localisation of the fluorescent signal, such as intensity of afluorescent signal in a specific cellular compartment above or below apredetermined threshold value. The selection criterion could also be apredetermined change in fluorescence lifetime or in fluorescencepolarization. Fluorescence intensity and/or localisation may for examplebe determined using image processing and/or image analysis, afluorescence microscope, FACS, FABS or fluorescence plate reader.

In one embodiment of the invention the selection criterion is highfluorescence intensity. This may for example be the case, when thecellular response is activation of a signal transduction pathway and thereporter system comprises a gene encoding a fluorescent protein, whereactivation of the signal transduction pathway leads to increasedexpression of said gene. Then resin beads may be selected using a methodcomprising the steps of:

-   1. Determining the fluorescence intensity of positive control resin    beads and setting this fluorescence intensity to 100%-   2. Determining the fluorescence intensity of negative control resin    beads and setting this fluorescence intensity to 0%-   3. Selecting resin beads having a fluorescence intensity    corresponding to at least 5%, preferably at least 10%, more    preferably at least 20%, even more preferably at least 30%, such as    at least 40%, for example at least 50%, such as at least 60%, for    example at least 70&, such as at least 80%, for example at least    90%, such as in the range of 5 to 100%, for example in the range of    10 to 100%, such as in the range of 20 to 100%, for example in the    range of 30 to 100%, such as in the range of 40 to 100%, for example    in the range of 50 to 100%.

The positive control may for example be a resin bead (or optionallyseveral resin beads kept in a separate container or well) comprising acompound known to influence the cellular response. By way of example, ifthe cellular response is activation of a signal transduction pathwaythrough a cell surface receptor, then the positive control may be aresin bead comprising a known ligand of said receptor, for example anaturally occurring ligand. The negative control may be a resin bead (oroptionally several resin beads kept in a separate container or well)optionally comprising a cell adhesion compound, but otherwise comprisingno library member or other test compound.

In another embodiment of the selection criterion is low fluorescence.This may for example be the case, when the cellular response isinhibition of a signal transduction pathway and the reporter systemcomprises a gene encoding a fluorescent protein, where an active signaltransduction pathway leads to expression of said gene. Then resin beadsmay be selected using a method comprising the steps of:

-   1. Determining the fluorescence intensity of positive control resin    beads and setting this fluorescence intensity to 0%-   2. Determining the fluorescence intensity of negative control resin    beads and setting this fluorescence intensity to 100%-   3. Selecting resin beads having a fluorescence intensity    corresponding to at least 5%, preferably at least 10%, more    preferably at least 20%, even more preferably at least 30%, such as    at least 40%, for example at least 50%, such as at least 60%, for    example at least 70&, such as at least 80%, for example at least    90%, such as in the range of 5 to 100%, for example in the range of    10 to 100%, such as in the range of 20 to 100%, for example in the    range of 30 to 100%, such as in the range of 40 to 100%, for example    in the range of 50 to 100%.

The positive control may for example be a resin bead (or resin beads)comprising a compound known to influence the cellular response. By wayof example, if the cellular response is inhibition of a signaltransduction pathway through a cell surface receptor, then the positivecontrol may be a resin bead comprising a known antagonist of saidreceptor. The negative control may be a resin bead (or resin beads)optionally comprising a cell adhesion compound, but otherwise comprisingno library member or other test compound.

One method of selecting resin beads using FABS is illustrated in FIG.1A.

In one preferred embodiment selection is performed manually with the aidof a fluorescence microscope. In this embodiment the fluorescenceintensity or other fluorescence properties are judged manually.

When the selection criterion is fluorescence intensity of localisation,the resin beads may also be analysed using a plate reader or imageacquisition. An example of such an analysis is given in FIG. 1B.

If the selection criterion is localisation, then resin beads aregenerally analysed by a fluorescence or imaging microscope. Saidmicroscope may optionally be equipped with a micromanipulator capable ofpicking out single beads. Resin beads are scanned for cells where thefluorescence signal is located at the desired intracellular location andthese resin beads are selected. The selection may be manually or it maybe automated.

In embodiments of the invention, wherein the detectable output is lightemission or the detectable output may be linked to a light signal, thenthe predetermined selection criterion could be any property of thelight. For example the selection criterion could be light intensityabove or below a predetermined threshold value. Light can be detectedfor example by the eye, in a microscope, and if the light is emitted viabioluminescence it can be measured by a luminometer.

In embodiments of the invention, wherein the detectable output is aradioactive signal or the detectable output may be linked to aradioactive signal, then the selection criterion could be any propertyof said radioactive signal, such as intensity above or below apredetermined threshold value or localisation of the radioactive signal.

In embodiments of the invention, wherein the detectable output is acolour signal or the detectable output may be linked to a colour signal,then the selection criterion could be any property or said coloursignal. For example the predetermined selection criterion could be acolour intensity above or below a specific threshold value or it couldbe a specific colour. The colour signal could be detected using anysuitable colorimetric method, such as a spectrophotometer,

Resin beads comprising cells meeting at least one selection criterion,such as any of the selection criteria mentioned herein above areselected. In certain embodiments of the invention resin beads comprisingcells meeting at least two, for example 2, such as 3, for example 4,such as in the range of 5 to 10, for example of in the range of 10 to 25selection criteria are selected.

It is also possible within the present invention to select resin beadscomprising cells meeting one or more predetermined selection criteriaand subsequently to subject said beads to one or more additionalselection rounds, wherein resin beads comprising cells meeting one ormore additional selection criteria are selected.

Resin beads meeting said at least one predetermined selection criteriamay be selected by manually sorting for example with the aid of amicroscope, for example by sorting by fluorescence or by colour or bymorphology depending on the detectable output and the selectioncriterion. Positive beads may be picked directly under the microscope,such as under a fluorescence microscope for example manually or with theaid of a micromanipulator. Frequently, in the range of 100 to 1,000,000,for example in the range of 1000 to 100,000, such as in the range of5000 to 50,000 resin beads may be placed on a suitable surface, such asin a dish or on a coverglass and subsequently examined by microscopy.Alternatively, the sorting process may be automated with the use ofspecially designed, commercially available bead sorters (UnionBiometrica, Sommerville, Mass.) and detecting for example fluorescenceintensity (Meldal, 2002, Biopolymers, 66: 93-100). In general, resinbeads can be sorted at a rate of about 100 beads per second, or evenfaster depending on the equipment used and its reading capacity. A rangeof about 5-30 beads per second is generally used with known instruments.Slower rates may be used to increase accuracy, however any suitable ratemay be used with the present invention, such as much higher rates.Preferred, is a rate where only one resin bead passes through thedetector at a time. It is also comprised within the present invention toselect resin beads using a plate reader. In general in the range of 1 to1000, such as 10 to 500, for example 50 to 100 resin beads are placed ineach well of a multiter plate and analysed. Beads from positive wellsmay then be further examined.

In one embodiment of the invention resin beads may be selected bycomparing the detectable output, with the detectable output generated bycontrol resin beads, for example positive and/or negative control resinbeads. Positive control resin beads are beads comprising a compoundcapable of inducing the desired cellular response, whereas negativecontrol resin beads comprises no such compound. By way of example, ifthe cellular response is activation of a cell surface receptor with aknown natural ligand, the positive control resin bead may comprise saidligand, whereas the negative control resin bead comprises no compoundexcept optionally a cell adhesion compound.

If the detectable output is a quantifiable signal, then resin beads maybe selected, comprising cells where the detectable output is higher orlower than the detectable output from cells attached to the positive ornegative control resin bead. By way of example, if the detectable outputis fluorescence intensity, then resin beads comprising cells displayinga fluorescence intensity which is higher than the negative control andlower than the positive control could for example be selected.

Non-limiting examples of methods of selecting resin beads areillustrates in FIGS. 1 and 2.

Identification of Compound

Once a resin bead has been selected, the compound of said bead may beidentified. Preferably, only one resin bead is used at a time. Thus ifsaid resin bead only comprises one library member in one or more copies,then only one compound is identified at a time.

The process for identification of the library member depends on the typeof library used. For a library of primarily oligomeric compounds, thelibrary member can be analysed by Mass Spectroscopy (MS), particularlyif the library was synthesized in such a way that the synthetic historyof the compound is captured, for example, using a capping procedure togenerate fragments of the compound that differ in mass by one buildingblock (see, for example, Youngquist et al., 1995, J. Am. Chem. Soc.,117: 3900-06). This capping procedure is most efficient when the cap andthe building block are reacted at the same time. The capping agent canbe any class of compound that has at least one functional group incommon with the building block used to generate the oligomer, so thatboth the capping agent and the building block can react when added tothe resin in an appropriate ratio. Alternatively, the capping agent canhave two functional groups in common with the building block where oneof the groups in common, such as the group in the building block that isused for the elongation of the oligomer, is orthogonally protected. Forexample, in a synthesis of a peptide using the Fmoc strategy, thecapping agent could be the same as the building block but with a Bocgroup protecting the reactive amine instead of the Fmoc group (see St.Hilaire et al., 1998, J. Am. Chem. Soc., 120: 13312-13320). In anotherexample, if the building block is a protected haloamine, the cappingagent could be the corresponding alkylhalide.

Where the library is synthesized by parallel synthesis (a parallelarray), the compound can be identified simply by the knowledge of whatspecific reaction components were reacted in a particular compartment.The structure can be confirmed by cleavage of a small portion ofcompound from the solid support and analyzed using routine analyticalchemistry methods such as infrared (IR), nuclear magnetic resonance(NMR), mass spectroscopy (MS), and elemental analysis. For a descriptionof various analytical methods useful in combinatorial chemistry, see:Fitch, 1998-99, Mol. Divers., 4: 39-45; and Analytical Techniques inCombinatorial Chemistry, M. E. Swartz (Ed), 2000, Marcel Dekker: NewYork.

In a preferred embodiment however the library has been synthesised by asplit-mix approach where the precise structure of the compound of aspecific bead is unknown. In this embodiment, the library member can beidentified using a variety of methods. The compound may be cleaved offthe resin bead, and then analyzed using IR, MS, or NMR. If the libraryis attached to the resin bead by a cleavable linker, then the compoundcan be cleaved by cleaving said cleavable linker. For NMR analysis,larger beads containing approximately 5 nmoles of material arepreferably used for the acquisition of 1-dimensional (1-D) and2-dimensional (2-D) NMR spectra. Furthermore, these spectra can beattained using high-resolution MAS NMR (magic angle spinning nuclearmagnetic resonance) techniques. Alternatively, high resolution-MAS NMRspectra can be acquired while the ligand is still bound to the solidsupport, as described for example, in Gotfredsen et al., 2000, J. Chem.Soc., Perkin Trans., 1: 1167-71. The compound may also be identified byrelease of the compound and fragmentation by MS-MS in MALDI orelectrospray mode.

Frequently, resin beads used for library synthesis contain about 100 to500 pmoles of material, which is generally insufficient for directanalysis using NMR techniques. In such situations, the libraries can besynthesised with special encoding to facilitate identification of thelibrary member. For a review of encoding strategies employed incombinatorial chemistry see: Barnes et al., 2000, Curr. Opin. Chem.Biol., 4: 346-50. Most coding strategies include the parallel synthesisof the encoding molecule (for example, DNA, PNA, or peptide) along withthe library compounds. This strategy requires a well-planned, timeconsuming, orthogonal protecting group scheme. Furthermore, the encodingmolecule itself can sometimes influence the cell leading to falsepositives. Alternatively, the library members can be encoded usingradiofrequency tags or using optical encoding, such as quantum dotencoding, spherical encoding or distance encoding. These methodsalleviates the problem of false positives stemming from the coding tags,but is generally only useful for small libraries in aone-bead-one-compound system due to the sheer bulk of the radiofrequencytag. Alternatively, single beads can be analyzed in a non-destructivemanner using infra-red imaging. This method gives limited informationand while useful for pre-screening, is not recommended for conclusivestructural determination.

In a preferred embodiment of the invention the library member(s)comprised within selected resin beads are identified using massspectrometry (MS). MS can be used alone to identify the library member.The library member can be cleaved from the resin bead, the molecularmass determined, and subsequently fragmented into sub-species toconclusively determine the structure. MS-based methods of compoundidentification are useful in this invention, as they require very littlematerial, and can utilise pico- to femtomole amounts of compound.MS-based methods include for example QTOF MSMS, MSMS or QTOF LC/MSMS.

After identification of the compound it may be desirable to confirm theactivity of said compounds by further in vitro and/or in vivo assays.For example, resin beads comprising the identified compound andoptionally an adhesion compound may be synthesized and the cellularresponse confirmed. It is also possible to test identified compounds inin vitro assays in the absence of beads. Cells may for example be growndirectly in a tissue culture dish, flask or coverglass and theidentified compound can be added directly to the medium of said cells.If several reporter systems are available for the particular cellularresponse then preferably several different reporter assays may be testedin vitro, in order to identify very useful compounds. For example,induction of a signal transduction pathway by a G-protein coupledreceptor frequently involves internalization of the G-protein coupledreceptor as well as a transcriptional response. Reporter systems forboth internalization and transcription may thus be tested.

Multiplexing

The methods disclosed by the present invention may also be used inmultiplexing methods.

For example, the methods may be used to identify compounds modifying atleast two cellular responses, such as 2, for example 3, such as 4, forexample in the range of 5 to 10, such as in the range of 10 to 25cellular responses.

In such methods step c) of the method outlined above (see the section“Summary of the invention”) preferably involves screening resin beadsfor beads comprising cells meeting at least two, such as 2, for example3, such as 4, for example in the range of 5 to 10, such as in the rangeof 10 to 25 predetermined selection criteria, wherein each selectioncriterion is preferably related to a different detectable output.

In such a method more than one kind of cell may be attached to eachresin bead and the different cellular responses may be detected indifferent kinds of cells. For example, a first cell line comprising afirst reporter system linked to a first cellular response and a secondcell line comprising a second reporter system linked to a secondcellular response and optionally additional cell line(s) comprisingadditional reporter system(s) linked to additional cellular response(s)may all be attached to a single bead. Resin beads comprising cellsmeeting selection criteria linked to all the different reporter systemsmay then be selected.

Depending on the detectable outputs, said detectable output may bedetermined using any of the methods described herein above. In onepreferred embodiment at least two detectable outputs are fluorescentoutputs, preferably of different excitation and/or emmision. Thus resinbeads meeting said at least two selection criteria may be selected inone step using a FABS with at least 2 channels in both excitation andemmision. Similarly, more than two different fluorescent properties maybe selected for in an suitable FABS. The at least two detectable outputsmay be in the same cell line or they may be in different cell lines.

Examples of multiplexing methods are illustrated in FIGS. 2A and 2B.

EXAMPLES Example 1 Screening of Adhesion Peptide Library

Approx. 100 adhesion peptide library beads were mixed with 1×10E6 cells(BHK, CHO, U2OS, Hek) in each well of a Falcon 12 well plate using 2 mlgrowth medium. The adhesion peptide library was prepared using thegeneral method for coupling amino acids outlined in example 5 below andinvolved

-   -   Coupling HMBA linker to PEGA-resin    -   Coupling amino acid to HMBA linker    -   SPPS coupling

The library consisted of heptamers of D-amino acids. The peptide librarybeads were PEGA beads each coupled to a potential adhesion peptide. Thecells and beads were mixed gently every 15 min for 2 hrs. Supernatantwith non-attached cells were removed and new growth medium added.Cells/beads were incubating for another 16 hrs. (37° C., 5% CO₂).

Cell adhesive beads were identified using a microscope with 10×objective and positive beads were transferred to a filter paper (to suckoff medium). Peptides were identified by amino acid sequencing. Examplesof useful peptides are given in table 2.

Example 1a

Identification of an Adhesion Peptide with Low Absorption of FluorescentComponents from Growth Medium and High Adhesion Properties:

An adhesion D-amino peptide library was synthesized (500,000 members) asdescribed above in Example 1 and screened for low fluorescence/highadherence properties. This was done in 4 steps:

1) Selection of low fluorescent beads by Fluorescence Activated BeadSorting (FABS).

The 500,000 member adhesion peptide library was FABSorted and 150,000low fluorescent beads were isolated.

2) Selection of beads with good cell adhesion properties.

The 150,000 low fluorescent beads were incubated with GFP expressingU2OS cells followed by FABS sorting for high fluorescence (high celladhesion). 536 beads were isolated.

3) Identification and isolation of beads with high Hek293 cell adherenceproperties.

The 536 beads were cleared for U2OS cells and incubated with GFPexpressing Hek293 cells. 47 beads with high cell adhesion propertieswere isolated using a fluorescence microscope.

4) Sequence elucidation and re-synthesis of selected peptides.

22 peptides were sequenced and six of them were re-synthesized. Based onStructure-Activity of the six peptides, four additional ones (AP-7 andAP-10) were synthesized. The peptide defined by SEQ IS 35 showed thebest overall performance.

An example of a method of preparing a resin bead comprising a usefuladhesion peptide is described in example 5, section “Synthesis ofadhesion peptide”.

Example 2

This example describes preparation of resin beads comprisingHis-(D)phe-Arg-Trp. These beads are for example useful as positivecontrol for in methods for identification of compounds modulating acellular response mediated through the melanocortin 4 receptor (MC4R).The synthesis is shown in FIG. 4.

Synthesis of Ac-His-(D)phe-Arg-Trp-NH₂

An overview of the synthesis is given in FIG. 4A.

PEGA resin (35 mg, 0.056 mmol) was swollen in dry DMF (1 mL) and treatedwith Fmoc-Rink amide linker (90.65 mg, 0.168 mmol, 3 equiv) in presenceof TBTU (51.77 mg, 0.224 mmol, 2.88 equiv) and NEM (28.3 μL, 0.224 mmol,4 equiv). After 3 h at room temperature, the resin was washed with DMF(10×), MeOH (10×), DCM (10×) and dried in vacuo. The resin was negativeto Kaiser amine test and a quantitative reaction was observed bymeasuring the Fmoc group on the resin (5 mg) with 20% Piperidine/DMFsolution (8 mL) for 30 min at room temperature.

The resin was swollen in dry DMF (1 mL) and the Fmoc group was removedby 20% Piperidine/DMF (1 mL) for 20 min at room temperature. The resinwas washed with DMF (10×) and the amino acids Fmoc-Trp(Boc),Fmoc-Arg(Pmc), Fmoc-(D)Phe and Fmoc-His(Trt) (3 equiv) were attachedsuccessively in presence of TBTU (2.88 equiv) and NEM (4 equiv). Afterthe incorporation of all amino acids, the Fmoc protection was removed by20% piperidine in DMF (1 mL, 20 min) and the resin was washed with DMF(10×). The peptide on the resin was then acetylated withaceticanhydride/pyridine/DMF (2:4:4) (1 mL) and washed with DMF (10×),MeOH (10×), DCM (10×) and dried in vacuo. The peptide was cleaved fromthe resin by treating with a solution of TFA (90%), water (5%),ethanedithiol (2%), triisopropyl silane (2%) and thioanisole (1%) for 3h at room temperature. The resin was filtered off and washed with TFA(2×) and DCM (2×). The combined filtrate was concentrated under vacuumand the peptide was precipitated by ether. The peptide was washed withether (10×) and dried in vacuo to afford 36.93 mg (96%) of pure peptide.

HPLC: t_(R)=9.61 min.

ESI-MS: calcd (M+H)⁺=686.78 Da; found (M+H)⁺=686.4

MALDI TOF MS: calcd (M+H)⁺=686.78 Da; found (M+H)⁺=686.98

¹H NMR (600 MHz, MeOH-d₄): δ=1.38-1.64 (m, 2H, Arg H^(β)), 1.10-1.15 (m,2H, Arg H^(γ)), 2.00 (s, 3H, Acetyl CH₃), 2.96 (m, 2H Arg H^(δ)),3.00-3.09 (m, 2H Phe H^(β)), 3.24-3.41 (m, 2H Trp H^(β)), 3.04-3.23 (m,2H His H^(β)), 4.01 (m, 1H Arg H^(α)), 4.73 (m, 1H His H^(α)), 4.51 (m,1H Phe H^(α)), 4.71 (m, 1H Trp H^(α)), 7.04-7.67 (br 5H Trp ringprotons), 7.21, 8.76 (2H, His ring protons), 7.25-7.33 (br, 5H Phe ringprotons).

Another compound useful as positive control in methods foridentification of compounds modulating a cellular response mediatedthrough the melanocortin 4 receptor (MC4R) is alfa-MSH of the sequenceAc-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-OH.

Synthesis of Ac-His-(D)phe-Arg-Trp-Gly-PEGA₁₉₀₀

These resin beads were used as positive controls in some of the belowmentioned examples.

An overview of the synthesis is given in FIG. 4B.

PEGA₁₉₀₀ library resin (100 mg, 0.02 mmol) was swollen in dry DMF (3 mL)and treated with Fmoc-Gly (17.84 mg, 0.06 mmol, 3 equiv) in presence ofTBTU (18.5 mg, 0.058 mmol, 2.88 equiv) and NEM (10.2 μL, 0.08 mmol, 4equiv). After 3 h at room temperature, the resin was washed with DMF(10×), MeOH (10×), DCM (10×) and dried in vacuo. The resin was negativeto Kaiser amine test and a quantitative reaction was observed bymeasuring the Fmoc group on the resin (5 mg) with 20% Piperidine/DMFsolution (8 mL) for 30 min at room temperature. The PEGA1900 libraryresin had previously been coupled to an adhesion peptide as described inExample 5a, in the section “Synthesis of adhesion peptide”.

The resin was swollen in dry DMF (1 mL) and the Fmoc group was removedby 20% Piperidine/DMF (1 mL) for 20 min at room temperature. The resinwas washed with DMF (10×) and the amino acids Fmoc-Trp(Boc),Fmoc-Arg(Pmc), Fmoc-(D)Phe and Fmoc-His(Trt) (3 equiv) were attachedsuccessively in presence of TBTU (2.88 equiv) and NEM (4 equiv). Afterthe incorporation of all amino acids, the Fmoc protection was removed by20% piperidine in DMF (1 mL, 20 min) and the resin was washed with DMF(10×). The peptide on the resin was then acetylated withaceticanhydride/pyridine/DMF (2:4:4) (1 mL) and washed with DMF (10×),MeOH (10×), DCM (10×) and dried in vacuo. The side chain protection ofthe peptide was removed by treating with a solution of TFA (90%), water(5%), ethanedithiol (2%), tri-isopropyl silane (2%) and thioanisole (1%)for 3 h at room temperature and the resin was washed with DCM (10×), DMF(10×) and water (10×).

Synthesis of Fmoc-Dap(N₃)OH

An overview of the synthesis is given in FIG. 4C

Fmoc-Dap-OH (980 mg, 3 mmol) was dissolved in 80% aqueous acetic acid (9mL) and CuSO₄.5H₂O (15 mg, 0.06 mmol, 0.02 equiv) in water (1 mL) wasadded. The pH of the solution was adjusted to 9-10 with K₂CO₃. Water (15mL), MeOH (32 mL) and trifluoromethanesulfonyl azide (6 mmol) in DCM (25mL) was added and the pH was readjusted to 9-10 with K₂CO₃. Thetwo-phase system was stirred vigorously for 20 h. The layers wereseparated by addition of DCM and the organic phase was washed with water(2×40 mL) and then the combined aqueous phases were acidified with 3 MHCl (aqueous) to a pH 2. The aqueous phase was extracted with DCM (4×50mL) and the combined organic phases were dried over sodium sulfate,filtered and concentrated under vacuo (0.934 g, 88.2%).

HPLC: t_(R)=10.08 min

ESI-MS: calcd (M+H)⁺=353.34 Da; found (M+H)⁺=353.1

¹H NMR (250 MHz, CDCl₃): δ=3.75 (d, 2H), 4.14-4.9 (t, 1H), 4.36-4.39 (d,2H), 4.50-4.54 (m, 1H), 5.50-5.54 (2H, NH and OH), 7.22-7.28 (4H,aromatic ring), 7.51-7.54 (d, 2H, aromatic ring), 7.68-7.71 (d, 2H,aromatic ring).

Example 3

This example describes synthesis of resin beads comprising a cycliccompound, which is capable of activating for example the melanocortin 4receptor. An overview of the synthesis is given in FIG. 5A.

Synthesis ofFmoc-Lys(Boc)-Dap(N₃)-His(Trt)-(D)phe-Arg(Pmc)-Trp(Boc)-Pra-Met-HMBA-Gly-PEGA

PEGA-red-resin (150 mg, 0.24 mmol) was swollen in dry DMF (5 mL) andtreated with Fmoc-Gly (215 mg, 0.72 mmol, 3 equiv) in presence of TBTU(222 mg, 0.69 mmol, 2.88 equiv) and NEM (121.8 μL, 0.96 mmol, 4 equiv).After 3 h at room temperature, the resin was washed with DMF (10×), MeOH(10×), DCM (10×) and dried in vacuo. The resin was negative to Kaiseramine test and a quantitative reaction was observed by measuring theFmoc group on the resin (5 mg) with 20% piperidine/DMF solution (8 mL)for 30 min at room temperature.

The resin was swollen in dry DMF (5 mL), Fmoc group was removed by 20%Piperidine/DMF and treated with HMBA linker (109.5 mg, 0.72 mmol, 3equiv) in presence of TBTU (222 mg, 0.69 mmol, 2.88 equiv) and NEM(121.8 μL, 0.96 mmol, 4 equiv). After 3 h at room temperature, the resinwas washed with DMF (10×), MeOH (10×), DCM (10×) and dried in vacuo. Theresin was negative to Kaiser amine test

The resin was swollen in dry DCM (2 mL), Fmoc-Met (267.5 mg, 0.72 mmol,3 equiv), MSNT (213.4 mg, 0.72 mmol, 3 equiv) and MeIm (43 μL, 0.54mmol, 2.25 equiv) were added. After 1 h, the resin was filtered andwashed with DCM (10×), MeOH (10×) and DMF (10×). The Fmoc group wasremoved by 20% Piperidine/DMF (1 mL) for 20 min at room temperature. Theresin was washed with DMF (10×) and the amino acids Fmoc-Pra,Fmoc-Trp(Boc), Fmoc-Arg(Pmc), Fmoc-(D)Phe, Fmoc-His(Trt), Fmoc-Dap(N₃)and Fmoc-Lys(Boc) (3 equiv) were attached successively in presence ofTBTU (2.88 equiv) and NEM (4 equiv). After the incorporation of allamino acids, the resin was washed with DMF (10×), MeOH (10×), DCM (10×)and dried in vacuo.

Cyclisation ofFmoc-Lys(Boc)-Dap(N₃)-His(Trt)-(D)Phe-Arg(Pmc)-Trp(Boc)-Pra-Met-HMBA-Gly-PEGA

-   a. The peptidyl resin (20 mg) was treated with a solution of TFA    (90%), water (5%), ethanedithiol (2%), triisopropyl silane (2%) and    thioanisole (1%) for 3 h at room temperature for removing all the    side chain protection groups. The resin was washed with DCM (10×),    MeOH (10×) and DMF (10×). The Fmoc group was removed by 20%    Piperidine/DMF (2 mL) and the resin was washed with DMF (10×), MeOH    (10×), DCM (10×) and THF (10×). DIPEA (61 μL, 0.35 mmol, 50 equiv)    and CuI (2.66 mg, 0.014 mmol, 2 equiv) in THF (300 μL) were added to    the resin. The reaction was left for 16 h and then washed with THF,    water, DMF, MeOH, DCM and dried in vacuo.-   b. The peptidyl resin (20 mg) was treated with DIPEA (61 μL, 0.35    mmol, 50 equiv) and CuI (2.66 mg, 0.014 mmol, 2 equiv) in THF (300    μL) were added to the resin. The reaction was left for 16 h and then    washed with THF, water, DMF, MeOH, DCM and dried in vacuo.-   c. Deprotection of the cyclic peptide. A solution of TFA (90%),    water (5%), ethanedithiol (2%), triisopropyl silane (2%) and    thioanisole (1%) were added to the resin for removing all the side    chain protection groups (3 h at room temperature). The resin was    washed with DCM (10×), MeOH (10×) and DMF (10×). The Fmoc group was    removed by 20% Piperidine/DMF (2 mL) and the resin was washed with    DMF (10×), MeOH (10×), DCM (10×) and dried in vacuo.    Cleavage of Peptide from the Resin

The resin was treated with 0.1 M NaOH (100 μL) for 2 h at roomtemperature. The resin was filtered and the filtrate was neutralisedwith 0.1 M HCl (100 μL).

(a) Yield=8.1 mg (82.5%)

(b) Yield=7.8 mg (79%)

HPLC: t_(R)=10.89 min

ES MS/MS: calcd (M+H)⁺=1112.29 Da; found (M+H)⁺=1112.56 Da

¹H NMR (600 MHz, DMSO-d₆): 1.259-1.273 (m, 2H Arg H^(γ)), 1.311-1.332(m, 2H Lys H^(γ)), 1.508-1.514 (m, 2H Lys H^(δ)), 1.416-1.616 (m, 2H ArgH^(β)), 1.650-1.669 (m, 2H Lys H^(β)), 1.852-1.979 (m, 2H Met H^(β)),2.022 (s, 3H Met —CH₃), 2.461 (t, 2H Met H^(γ)), 2.484-2.577 (m, 2H PraH^(β)), 2.671-2.848 (m, 2H His H^(β)), 2.721-2.944 (m, 2H Phe H^(β)),2.728-2.734 (t, 2H Lys H^(ε)), 2.961-3.157 (m, 2H Trp H^(β)),2.998-3.004 (m, 2H Arg H^(δ)), 3.366-3.568 (m, 2H Dap H^(β)), 3.794 (m,1H Lys H^(α)), 4.282 (m, 1H Arg H^(α)), 4.309 (m, 1H Met H^(α)), 4.417(m, 1H Pra H^(α)), 4.521 (m, 1H Dap H^(α)), 4.568 (m, 1H Trp H^(α)),4.584 (m, 1H His H^(α)), 4.662 (m, 1H Phe H^(α)), 7.164-7.239 (br, 5HPhe ring protons), 7.201, 8.211 (2H, His ring protons), 7.447 (s, 1H Arg—NH), 6.955-7.312 (br, 5H Trp ring protons), 8.240 (s, 1H Triazole ringproton), 8.094 (1H Trp amide H), 8.185 (1H Met amide H), 8.213 (1H Pheamide H), 8.250 (1H Pra amide H), 8.329 (1H Arg amide H), 8.408 (1H Hisamide H), 8.805 (1H Dap amide H), 10.697 (1H Trp ring NH).

Example 4 Synthesis of Cyclic Peptide Library

The cyclic peptide library of example 4 is for example useful foridentification of compounds capable of modulating a cellular responsemediated through the melacortin 4 receptor.

Fmoc-Lys(Boc)-Dap(N₃)-Aa1-Aa2-Aa3-Aa4-Pra-Met-HMBA-Gly-PEGA-NH-Gly-Alloc

PEGA resin (1.5 g, 0.3 mmol) is swollen in dry DMF (15 mL) and treatedwith a mixture of Fmoc-Gly (268 mg, 0.9 mmol, 3 equiv) and Alloc-Gly(143 mg, 0.9 mmol, 3 equiv) by preactivation with TBTU (277 mg, 0.86mmol, 2.88 equiv) and NEM (152 μL, 1.2 mmol, 4 equiv) and slow additionof the activated mixture to the resin. After 3 h at room temperature,the resin is washed with DMF (10×), MeOH (10×), DCM (10×) and dried invacuo. The resin is negative to Kaiser amine test and a 1:2 ratio ofAlloc:Fmoc is observed by measuring the Fmoc group on the resin (5 mg)with 20% Piperidine/DMF solution (15 mL) for 30 min at room temperatureand determination of the absorption of the eluate at 305 nm.

The resin is swollen in dry DMF (15 mL), Fmoc group is removed by 20%Piperidine/DMF and treated with HMBA linker (92 mg, 0.6 mmol, 3 equiv)in presence of TBTU (185 mg, 0.58 mmol, 2.88 equiv) and NEM (102 μL, 0.8mmol, 4 equiv). After 3 h at room temperature, the resin is washed withDMF (10×), MeOH (10×), DCM (10×) and dried in vacuo. The resin isnegative to Kaiser amine test.

The resin is swollen in dry DCM (20 mL), Fmoc-Met-OH (223 mg, 0.6 mmol,3 equiv), MSNT (178 mg, 0.6 mmol, 3 equiv) and MeIm (36 μL, 0.45 mmol,2.25 equiv) were added. After 1 h, the resin is filtered and washed withDCM (10×), MeOH (10×) and DMF (10×). The Fmoc group is removed by 20%Piperidine/DMF (15 mL) for 20 min at room temperature. The resin iswashed with DMF (10×) and Fmoc-Pra is attached to the resin in presenceof TBTU and NEM. The resin is transferred to a 20 well multiple columnpeptide synthesiser and distributed equally in to each wells. Aminoacids Fmoc-Aa4-OH, Fmoc-Aa3-OH, Fmoc-Aa2-OH, Fmoc-Aa1-OH, Fmoc-Dap(N₃)and Fmoc-Lys(Boc) (3 equiv) are attached successively in presence ofTBTU (2.88 equiv) and NEM (4 equiv). After the incorporation of allamino acids, the resin is washed with DMF (10×), MeOH (10×), DCM (10×)and dried in vacuo.

The synthesis is illustrated in FIG. 5B.

Fmoc-NH—CH(R₁)—CO may be any natural amino acids coupled to Fmoc

Fmoc-NH—CH(R₂)—CO; Fmoc-NH—CH(R₃)—CO; Fmoc-NH—CH(R₄)—CO may be any ofthe following amino acids coupled to Fmoc: Cys, Phe, His, Lys, Met, Pro,Arg, Ser, Thr, Val, Trp, Tyr, Homophenyl alanine, Tic, 4-Phenylpyrrolidone 2-carboxylic acid, 1-Aminocyclohexane carboxylic acid,4-Pyridyl alanine, (D)-Orn Hyp, 4-Phenyl peperidine carboxylic acid.

Cyclisation ofFmoc-Lys(Boc)-Dap(N₃)-Aa1-Aa2-Aa3-Aa4-Pra-Met-HMBA-Gly-PEGA-NH-Alloc

The peptidyl resin is treated with a solution of TFA (90%), water (5%),ethanedithiol (2%), triisopropyl silane (2%) and thioanisole (1%) for 3h at room temperature for removing all the side chain protection groups.The resin is washed with DCM (10×), MeOH (10×) and DMF (10×). The Fmocgroup is removed by 20% piperidine/DMF (15 mL) and the resin is washedwith DMF (10×), MeOH (10×), DCM (10×) and THF (10×). DIPEA (1.75 mL, 10mmol, 50 equiv) and CuI (76.2 mg, 0.4 mmol, 2 equiv) in THF (10 mL) areadded to the resin. The reaction is left for 16 h and then washed withTHF, water, DMF, MeOH, DCM and dried in vacuo.

The cyclisation process is illustrated in FIG. 5B

Synthesis of Adhesion Peptide ((D)Arg-(D)Arg-(D)Ile-(D)Arg-Gly) onCyclic Peptide Library Beads

a. Alloc Deprotection

-   -   Pd(PPh₃)₄ (346.5 mg, 0.3 mmol, 3 equiv) is dissolved in acetic        acid (5%) and NEM (2.5%) in chloroform (15 mL) and degassed by        purging with Ar for 10 min. The reaction mixture is added to the        lyophilised resin under Ar atmosphere and kept for 20 min at        room temperature.        b. Synthesis of Adhesion Peptide    -   The resin is washed with DMF (10×) and Fmoc-Lys(Boc) (141 mg,        0.3 mmol, 3 equiv) is attached using TBTU (92 mg, 0.288 mmol,        2.88 equiv) and NEM (51 μL, 0.4 mmol, 4 equiv). The resin is        washed with DMF (10×) and the α-Fmoc and side chain Boc        protections are removed by 20% piperidine (15 mL) and 30% TFA in        DCM (20 mL) respectively. The resin is again treated with        Fmoc-Lys(Boc) (282 mg, 0.6 mmol, 3 equiv) and TBTU (184 mg,        0.576 mmol, 2.88 equiv) and the Fmoc and Boc protections are        removed by 20% piperidine in DMF and 30% TFA in DCM. The resin        is washed with DMF (10×) and two residues of Ahx are attached by        adding TBTU activated Fmoc-Ahx (425 mg, 1.2 mmol, 3 equiv). The        amino acids Fmoc-Gly, Fmoc-(D) Arg(Pmc) and Fmoc-(D) Ile (3        equiv) are attached according to the sequence in presence of        TBTU (2.88 equiv) and NEM (4 equiv). After the incorporation of        all amino acids, the N-terminal Fmoc group is removed by 20%        piperidine in DMF (15 mL) and the resin is washed with DMF        (10×), MeOH (10×), DCM (10×) and dried in vacuo.    -   The peptidyl resin is treated with a solution of TFA (90%),        water (5%), ethanedithiol (2%), triisopropyl silane (2%) and        thioanisole (1%) for 3 h at room temperature for removing all        the side chain protection groups. The resin is washed with DCM        (10×), MeOH (10×) and DCM (10×) and dried in vacuo.

Example 5 Library of Oligocyclic Ureas as Peptidomimetics.

This library is for example useful for identification of compoundsmodulating a cellular response mediated through a G-protein coupledreceptor.

Synthesising Combinatorial Library of Potential Urea GPCR Agonist ViaSPPS and the Pictet-Spengler Reaction: Experimental: General:

All chemicals described, apart from the building block O-Pfp carbamatesare commercially available and used without further purification. Thebuilding block O-Pfp carbamates are prepared as described in: Diness,F.; Beyer, J.; Meldal, M.; J. Combi. Chem. and QSAR. 2004, 23, 1-15. Allsolvents are HPLC-grade. PEGA₉₀₀-resin is purchased from VersaMatrixA/S. Each washing step lasts 2 min unless otherwise stated.

Coupling of HMBA Linker to PEGA₉₀₀-Resin:

Dry PEGA₉₀₀-resin is swelled in DCM and washed with DMF (3×). 3.0 eq.HMBA, 2.9 eq. TBTU and 3.0 eq. NEM are mixed in appropriate DMF andallowed to react for 10 min. The mixture is added to resin and after 2 hthe resin is washed with DMF (6×), DCM (6×) and lyophilised.

General Procedure for Coupling of Amino Acid to HMBA-Linker

Dry PEGA₉₀₀-resin with HMBA-linker is swelled in DCM. 3.0 eq.Fmoc-protected amino acid, 2.25 eq. MeIm and 3.0 eq. MSNT are mixed inappropriate amount of DCM and added to resin. After 1 h the resin iswashed with DCM (3×) and the coupling is repeated as above once. Aftercoupling for 1 h the resin is washed with DCM (6×), DMF (6×), DCM (6×)and lyophilised.

General SPPS Coupling Procedure

The terminal amino acid on the resin is Fmoc-deprotected by treatmentwith 20% piperidine in DMF (1×2 min+1×18 min) followed by washing withDMF (6×). 3.0 eq. Fmoc-protected amino acid, 2.9 eq. TBTU and 3.0 eq.NEM are mixed in appropriate amount of DMF and allowed to react for 10min. The mixture is added to the resin and after 2 h the resin is washedwith DMF (6×).

General Building Block Coupling Procedure

The terminal amino acid on the PEGA₉₀₀-resin with HMBA linker andtetrapeptide is Fmoc-deprotected by treatment with 20% piperidine in DMF(1×2 min+1×17 min) followed by washing with DMF (6×). 3.0 eq. buildingblock —O-Pfp carbamate is dissolved in appropriate amount of DMF and thesolution is added to resin. After ended coupling the resin is washedwith DMF (6×), DCM (6×) and lyophilised.

General Pictet-Spengler Reaction Procedure

Dry PEGA₉₀₀-resin with HMBA linker, peptide and building block isswelled in 10% TFA (aq) (1×1 h and 1×11 h). The resin is washed with H₂Ountil washing water has pH=6-7 and washed with DMF (6×), DCM (6×) andlyophilised.

General Side Chain Deprotection Procedure

Dry PEGA₉₀₀-resin with HMBA linker, peptide and Pictet-Spengler productis swelled in H₂O and the side chains are deprotected with 95% TFA (aq)(2×15 min). The resin is washed with H₂O until washing water had pH=5-7.The resin is then washed with DMF (6×), DCM (6×) and lyophilised.

General HMBA Cleavage Procedure

Dry PEGA₉₀₀-resin with HMBA linker and attached compound is swelled inwater and NaOH (aq.) 0.1 M is added. After 2 h HCl (aq.) 0.1 M is usedfor neutralisation and then AcN was added until the H₂O/AcN ratio is 1:1by volume. The resin is filtered off and the liquid is used direct forRP-HPLC or/and Q-TOF MS analysis.

Synthesising the Combinatorial Library:

Dry PEGA₉₀₀-resin (1.0 g, 0.2 mmol) is coupled with HMBA linker asdescribed in “Coupling of HMBA linker to PEGA₉₀₀-resin” an equimolarmixture of Fmoc-glycine and Alloc glycine is the coupled to the HMBAfunctionalised PEGA₉₀₀-resin as described in “General Procedure forCoupling of amino acid to HMBA linker”. An analytical sample is cleavedby “General HMBA Cleavage Procedure” and tested by RP-HPLC. TheFmoc-Gly-HMBA-PEGA₉₀₀-resin is swelled in DCM, washed with DMF (6×) anddivided into the wells of a 20-welled peptide synthesiser. The 10different amino acids are coupled to the glycine using “General SPPSCoupling Procedure”. The resin from all the wells is mixed again anddivided into the wells of a 20-welled peptide synthesiser and the 10different amino acids are coupled to the terminal amino acid using“General SPPS Coupling Procedure”. The resins from all the wells aremixed again and divided into the wells of a 20-welled peptidesynthesiser and the 10 different trypthophan derivatives acids arecoupled to the terminal amino acid using “General SPPS CouplingProcedure”. The resins from all the wells is mixed again and dividedinto the wells of a 20-welled peptide synthesiser and the 10 differentbuilding blocks are coupled to the terminal amino acid using “GeneralBuilding Block Coupling Procedure”. The resin from all the wells ismixed again and the Pictet-Spengler reaction is performed as describedin “General Pictet-Spengler Reaction Procedure”. The Alloc group isremoved from amino groups with 5 mol % Pd(P(Ph₃))₄ in DMF containing 1%morpholinium acetate. Boc/tBu/Pcm protected adhesion peptide 4 (2 eqv)is coupled using TBTU/NEM preactivation (5 min, 0° C.) for 14 h, untilKaiser test showed complete reaction. This is followed by Boc-, Bu^(t)and Pmc-deprotection as described in “General Side Chain DeprotectionProcedure”. Finally analytical samples are cleaved from single beads by“General HMBA Cleavage Procedure” and tested by Q-TOF MS and MSMSanalysis.

The structure of the resulting library members is given below.

R₁=dipeptide. Any combination of Gly, L-Trp, L-Arg, D-Arg, L-His, L-Phe,D-Phe, L-Lys, L-Asn, 4-amino-L-Phe

R₂=H, 5-OH, 5-Br, 6-F, 7-N₃, 5-OMe

R₁=dipeptide. Any combination of Gly, L-Trp, L-Arg, D-Arg, L-His, L-Phe,D-Phe, L-Lys, L-Asn, 4-amino-L-Phe

R₂=H, 5-OH, 5-Br, 6-F, 7-N₃, 5-OMe R₃=R₄=Me

orR₃=H and R₄=H, iPr, H₂N—CH₂, Ph-CH₂, (4-HO—)Ph-CH₂ or indo-2-ly-CH₂or

R₃=Phe and R₄=H

iPr=

H₂N—CH₂=

Ph-CH₂=

(4-HO—)Ph-CH₂=

indo-2-ly-CH₂=

Example 5a Library of Oligocyclic Ureas as Peptidomimetics 2.

This library is for example useful for identification of compoundsmodulating a cellular response mediated through a G-protein coupledreceptor.

Synthesising Combinatorial Library of Potential Urea GPCR Agonist ViaSPPS and the Pictet-Spengler Reaction: Experimental: General:

All chemicals described, apart from the building block O-Pfp carbamatesare commercially available and used without further purification. Thebuilding block O-Pfp carbamates are prepared as described in: Diness,F.; Beyer, J.; Meldal, M.; J. Combi. Chem. and QSAR. 2004, 23, 1-15. Allsolvents are HPLC-grade. PEGA₉₀₀-resin is purchased from VersaMatrixA/S, Denmark. Each washing step lasts 2 min unless otherwise stated.

General SPPS Coupling Procedure

The terminal amino acid on the resin is Fmoc-deprotected by treatmentwith 20% piperidine in DMF (1×2 min+1×18 min) followed by washing withDMF (6×). 3.0 eq. Fmoc-protected amino acid or HMBA, 2.9 eq. TBTU and4.0 eq. NEM are mixed in appropriate amount of DMF and allowed to reactfor 10 min. The mixture is added to the resin and after 2 h the resin iswashed with DMF (6×).

General Procedure for Coupling of Amino Acid to HMBA-Linker

Dry PEGA₁₉₀₀-resin with HMBA-linker is swelled in DCM. 3.0 eq.Fmoc-protected amino acid, 2.25 eq. MeIm and 3.0 eq. MSNT are mixed inappropriate amount of DCM and added to resin. After 1 h the resin iswashed with DCM (3×) and the coupling is repeated as above once. Aftercoupling for 1 h the resin is washed with DCM (6×), DMF (6×), DCM (6×)and lyophilised.

General Building Block Coupling Procedure

The terminal amino acid on the PEGA₁₉₀₀-resin with HMBA linker andtetrapeptide is Fmoc-deprotected by treatment with 20% piperidine in DMF(1×2 min+1×17 min) followed by washing with DMF (6×). 3.0 eq. buildingblock —O-Pfp carbamate is dissolved in appropriate amount of DMF and thesolution is added to resin. After ended coupling the resin is washedwith DMF (6×), DCM (6×) and lyophilised.

General Pictet-Spengler Reaction Procedure

Dry PEGA₉₀₀-resin with HMBA linker, peptide and building block isswelled in 10% TFA (aq) (1×1 h and 1×11 h). The resin is washed with H₂Ountil washing water has pH=6-7 and washed with DMF (6×), DCM (6×) andlyophilised.

General Side Chain Deprotection Procedure

Dry PEGA₁₉₀₀-resin with HMBA linker and attached compounds is swelled inH₂O and the side chains are deprotected with 95% TFA (aq) (2×15 min).The resin is washed with H₂O until washing water had pH=5-7. The resinis then washed with DMF (6×), DCM (6×) and lyophilised.

General HMBA Cleavage Procedure

Dry PEGA₁₉₀₀-resin with HMBA linker and attached compounds is swelled inwater and NaOH (aq.) 0.1 M is added. After 2 h HCl (aq.) 0.1 M is usedfor neutralisation and then AcN was added until the H₂O/AcN ratio is 1:1by volume. The resin is filtered off and the liquid is used direct forRP-HPLC or/and Q-TOF MS analysis.

Synthesising the Combinatorial Library:

Dry PEGA₁₉₀₀-resin (1.0 g, 0.2 mmol) is coupled with an equimolarmixture of Fmoc-glycine and Alloc glycine as described in “General SPPSCoupling Procedure”. HMBA is coupled as described in “General SPPSCoupling Procedure”. Fmoc-glycine is the coupled to the HMBAfunctionalised PEGA₁₉₀₀-resin as described in “General Procedure forCoupling of amino acid to HMBA linker”. TheFmoc-Gly-HMBA-Gly-PEGA₁₉₀₀-Gly-Alloc resin is swelled in DCM, washedwith DMF (6×) and divided into the wells of a 20-welled peptidesynthesiser. The 20 different natural L-amino acids are coupled to theglycine using “General SPPS Coupling Procedure”. The resin from all thewells is mixed again and divided into the wells of a 20-welled peptidesynthesiser and the 20 different natural L-amino acids are coupled tothe terminal amino acid using “General SPPS Coupling Procedure”. Theresins from all the wells are mixed again and divided into 10 wells of a20-welled peptide synthesiser and the 10 different Fmoc-protectedtryptophane derivatives (shown in Table 6) are coupled to the terminalamino acid using “General SPPS Coupling Procedure”. The resins from allthe wells is mixed again and divided into 8 wells of a 20-welled peptidesynthesiser and the 8 different building blocks (shown in Table 6) arecoupled to the terminal amino acid using “General Building BlockCoupling Procedure”. The resin from each well is transferred into asyringe with a filter in the bottom. The Alloc group is removed from theresin bound glycine by using 5 mol % Pd(P(Ph₃))₄ in chloroformcontaining 5% AcOH and 2.5% NEM under argon for 12 h. The resin is thenwashed with chloroform (6×), 0.5% Et₂NCS₂Na-3H₂O and 0.5% DIPEA in DMF(6×) and DMF (10×). 1.5 eq. protected adhesion peptide (AP4), 1.4 eq.TBTU and 2.0 eq. NEM are mixed in appropriate amount of DMF and allowedto react for 10 min. The mixture is added to the resin and added toresin. When the Kaiser test shows complete reaction the resin is washedwith DMF (6×) and DCM (6×). The Pictet-Spengler reaction is performed asdescribed in “General Pictet-Spengler Reaction Procedure”. This isfollowed by side chain deprotection as described in “General Side ChainDeprotection Procedure”. Finally analytical samples are cleaved fromsingle beads by “General HMBA Cleavage Procedure” and tested by Q-TOF MSand MSMS analysis.

TABLE 6 Tryptophane derivatives Building Blocks

The structure of the resulting library members is given below.

R₁=dipeptide. Any combination of the 20 natural occurring L amino acids

R₂=H, 5-OH, 5-OMe, 5-OBn, 5-Br, 5-F, 6-F, 5-Me, 6-Me, 7-Me

R₃=H, iPr, H₂N—CH₂, Ph-CH₂, (4-HO—)Ph-CH₂ or indol-2-ly-CH₂iPr=

H₂N—CH₂=

Ph-CH₂=

(4-HO—)Ph-CH₂=

indo-2-ly-CH₂=

Synthesis of Protected Adhesion Peptide

PEGA₉₀₀-resin is swelled in DMF. 3.0 eq. HMBA, 2.9 eq. TBTU and 4.0 eq.NEM are mixed in appropriate amount of DMF and allowed to react for 5min. The mixture is added to resin and after 2 h the resin is washedwith DMF (6×), DCM (6×) and lyophilised. The resin is swelled in DCM and3.0 eq. Fmoc-Gly-OH, 2.25 eq. MeIm and 3.0 eq. MSNT are mixed inappropriate amount of DCM and added to resin. After 1 h the resin iswashed with DCM (3×) and the coupling is repeated as above once. Aftercoupling for 1 h the resin is washed with DCM (6×), DMF (6×), DCM (6×)and lyophilised. The resin is swelled in DMF and a sequence ofFmoc-gln(trt)-OH, Fmoc-arg(Pmc)-OH, Fmoc-ile-OH, Fmoc-arg(Pmc)-OH,Fmoc-lys(Boc)-OH and Boc-ala-OH is coupled as described in “General SPPSCoupling Procedure”. The resin is then washed with DMF (6×), DCM (6×)and lyophilised. The final peptide(Boc-ala-arg(Pmc)-lys(Boc)-arg(Pmc)-ile-arg(Pmc)-gln(trt)-GlyOH) iscleaved from the resin as described in “General HMBA CleavageProcedure”.

Example 6a Library of Multi-Heterocyclic Peptidomimetics for GPCRReceptors (Library 6a).

This library is for example useful for identification of compoundsmodulating a cellular response mediated through a G-protein coupledreceptor.

Library Design and Synthesis

All Pictet-Spengler reaction methodology has been developed and testedon the synthesis resin PEGA₈₀₀,¹ wherefore the analogous library resinPEGA₁₉₀₀ is chosen for the library synthesis. In order to screen foractive compounds, the library is prepared following a“one-bead-two-compounds” strategy. This is accomplished by treating theamino-functionalized resin with a mixture of Fmoc-Gly-OH:Alloc-Gly-OH(10:1) activated by the TBTU procedure² to provide orthogonal reactionsites for (a) split-and-mix library synthesis (via the Fmoc handle); and(b) attachment of an adhesion molecule (AM) (via the Alloc handle). Thelibrary synthesis of Pictet-Spengler reaction precursor 1 is carried outaccording to standard Fmoc amino acid coupling protocols for solid-phasepeptide synthesis (FIG. 6 a). Due to the requirement of acidic reactionconditions for the Pictet-Spengler reaction step (q), the base labileHMBA (hydroxymethylbenzoic acid) linker is employed. Prior to attachmentof HMBA to H₂N-Gly-PEGA₁₉₀₀ via the TBTU activation procedure, the Fmocprotecting group is removed by standard piperidine treatment. The HMBAlinker provides a convenient cleavage site for quantitative release fromthe solid support via basic hydrolysis. Cleavage of product from asingle bead is routinely achieved by treating the bead with 0.1 M NaOH(aq) overnight, thus providing amounts of material sufficient forstructure elucidation via QTOF ES-MSMS analysis. After splitting theresin portion into 10 different wells, the hydroxy handle of the linkeris esterified by treatment with 10 MSNT-activated Fmoc amino acids(Fmoc-AA₁-OH),³ thus attaching the first amino acid residue of thepeptidomimetic sequence. Subsequent analogous split-and-mix synthesisand 3 cycles of Fmoc deprotection/TBTU-mediated couplings of 10 Fmocamino acids as the second amino acid residue (Fmoc-AA₂-OH), 15 Fmocamino acids incorporating the reactive aromatic side-chain(Fmoc-AA₃-OH), and 7 masked aldehyde building blocks (R⁴-MABB-OH) (Table5a), prepared as previously reported,^(4,5) afford the Pictet-Spenglerreaction precursor 1. In this coupling sequence, one fifth of the resinis withdrawn prior to the coupling of Fmoc-AA₂-OH (steps e and f), andremixed with the remaining resin from step g and forth. Ultimately, thisaffords a library composed of tripeptoidal (n=0) and tetrapeptoidal(n=1) substructures. The Alloc protecting group of 1 is removed withPd(PPh₃)₄, and subsequent TBTU coupling of Fmoc-Lys(Fmoc)-OH/Fmocdeprotection (×2) provided the amino handles for attachment of theadhesion molecule AM, which is accomplished via the TBTU activationprocedure. The adhesion molecule is synthesized via standard solid-phasepeptide synthesis, and purified by preparative HPLC prior to attachmentto resin. To finalize the library synthesis, the resin 2 is treated with10% TFA (aq), which simultaneously facilitates the intramolecularN-acyliminium Pictet-Spengler reaction and removal of the Boc-protectinggroups in the side-chains of AA₁ (R¹) and AA₂ (R²). As a consequence ofthe structurally diverse aromatic heterocycles undergoing theintramolecular N-acyliminium Pictet-Spengler reaction, the library isgraphically represented by the six sublibraries (Ia-VIa) below (FIG. 6a). Theoretically, the library is composed by 11270 different cornpounds(32890 when all stereoisomers are counted).

An overview of the synthesis of a combinatorial library via theintramolecular N-acyliminium Pictet-Spengler reaction^(a,b) is given inFIG. 6 a. The amino acids and building blocks used for the librarysynthesis are indicated in table 5a.

Reagents and conditions: (a) Fmoc-Gly-OH:Alloc-Gly-OH (9:1), TBTU, NEM,DMF; (b) 20% piperidine (DMF); (c) HMBA, TBTU, NEM, DMF; (d)Fmoc-AA₁-OH, MSNT, MeIm, CH₂Cl₂; (e) 20% piperidine (DMF); (f)Fmoc-AA₂-OH, TBTU, NEM, DMF; (g) 20% piperidine (DMF); (h) Fmoc-AA₃-OH,TBTU, NEM, DMF; (i) 20% piperidine (DMF); (j) R⁴-MABB-OH, TBTU, NEM,DMF; (k) Pd(PPh₃)₄ (CHCl₃:AcOH:NEM (925:50:25); (l) Fmoc-Lys(Fmoc)-OH,TBTU, NEM, DMF; (m) 20% piperidine (DMF); (n) Fmoc-Lys(Fmoc)-OH, TBTU,NEM, DMF; (o) 20% piperidine (DMF); (p) AM-OH, TBTU, NEM, DMF; (q) 10%TFA (aq);^(a) Sublibraries Ia, IIIa, IVa, Va and VIa each consists of700 different compounds (1300 when all stereoisomers are counted) withn=1, and 70 different compounds (130 when all stereoisomers are counted)with n=0;^(b) Sublibrary IIa consists of 7000 different compounds (23400when all stereoisomers are counted) with n=1, and 700 differentcompounds (2340 when all stereoisomers are counted) with n=0.

TABLE 5a Amino acids and building blocks for combinatorial librarysynthesis

AA₁ AA₂ AA₃ (Sublibrary structure) R⁴ D-Phe Phe L-3,4-Dimethoxyphe (Ia)H D-Tyr(t-Bu) Tyr(t-Bu) Trp (IIa) Me D-Arg(Boc)₂ Arg(Boc)₂ D/L-(5-Br)Trp(IIa) i-Bu D-Lys(Boc) Lys(Boc) L-(5-OH)Trp (IIa) Bn D-His(Boc) His(Boc)D/L-(5-MeO)Trp (IIa) Ph D-Trp Trp D/L-(4-Me)Trp (IIa) 4-Br—PhL-(1-Np)Ala L-(1-Np)Ala D/L-(5-Me)Trp (IIa) 3-CF₃—Ph L-Homophe L-HomopheD/L-(6-Me)Trp (IIa) L-(3-CN)Phe L-(3-CN)Phe D/L-(5-BnO)Trp (IIa)L-(4-CF₃)Phe L-(4-CF₃)Phe D/L-(5-F)Trp (IIa) D/L-(6-F)Trp (IIa)L-(2-Thi)Ala (IIIa) L-(3-Thi)Ala (IVa) L-(2-Fur)Ala (Va) L-(3-BzThi)Ala(VIa)

General Methods. All solvents are of HPLC quality and stored overmolecular sieves. Solid-phase organic combinatorial chemistry isroutinely carried out using a 20-well peptide synthesizer equipped withsintered teflon filters (50 μm pores), teflon tubing, and valves, whichallow suction to be applied below the wells. For all reactions on solidsupport, PEGA₁₉₀₀ resin (0.2 mmol/g, VersaMatrix A/S) is used. Prior touse, the resin is washed with methanol (×6), DMF (×6), and CH₂Cl₂ (×6).All commercially available reagents are used as received without furtherpurification. Analysis of all solid-phase reactions is performed aftercleaving the products as their free acids from the resin. A single beadis treated with 0.1 M aqueous NaOH (10 μL) in a 0.5 mL Eppendorf tubeovernight, then diluted with CH₃CN (20 μL), before filtering thesolution, thereby providing a sample for ES MSMS analysis on a MicroMassQTOF Global Ultima mass spectrometer (mobile phase 50% CH₃CN (aq), 0.1μL/min) employing a linear ramping of the collision energy. Spectra(FIG. 7) are analyzed by generating the exact mass differences betweenfragment ions and tabulated (FIG. 8) to provide the fragmentationpathway (FIG. 9) and therefore structure of the compound released fromthe single bead.

Solid-Phase Synthesis of Combinatorial Library (6a).

Attachment of Fmoc-Gly-OH/Alloc-Gly-OH to the amino-functionalizedPEGA₁₉₀₀ resin (1.00 g) is carried out by premixing Fmoc-Gly-OH (0.62mmol, 185 mg):Alloc-Gly-OH (0.07 mmol, 9.9 mg) (9:1, 3.0 equiv intotal), N-ethyl morpholine (NEM, 0.92 mmol, 106 mg, 4.0 equiv), andN-[1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminiumtetrafluoroborate N-oxide (TBTU, 0.66 mmol, 213 mg, 0.88 equiv) for 5min in DMF. The resulting solution is added to the DMF preswollen resinand allowed to react for 5 h, followed by washing with DMF (×6), andCH₂Cl₂ (×6). Completion of the reaction is monitored using the Kaisertest. Prior to attachment of the HMBA linker via the procedure above,Fmoc-deprotection was accomplished with 20% piperidine in DMF, first for2 min, and then for 18 min, followed by washing with DMF (×6). Couplingof the first amino acid (Fmoc-AA₁-OH) to the HMBA derivatized resin isaccomplished by treating the freshly lyophilized resin, split in 20(2×10) wells via dry CH₂Cl₂, with a mixture of the Fmoc-AA₁-OH (4.5equiv), MeIm (3.4 equiv), and MSNT (4.5 equiv) in CH₂Cl₂:THF (5:1).³ Thecoupling is carried out for 1 h. When split in 20 wells, each well isassumed to hold ca. 50 mg resin, and accordingly added reagents relativeto 0.01 mmol of material on the solid phase. Excess reagents are removedwith suction below each well, followed by washing with dry DMF (×1), anddry CH₂Cl₂ (×1), before repeating the MSNT coupling of Fmoc-AA₁-OH once.Subsequent split-and-mix peptide syntheses with Fmoc-AA₂-OH,Fmoc-AA₃-OH, and R⁴-MABB-OH, respectively, are accomplished followingthe coupling procedure described above for the attachment of Fmoc-Gly-OH(via TBTU and NEM in DMF).² The usual washing protocol followed eachcoupling and deprotection step, and all couplings are checked via theKaiser test. The Alloc group of 1 is removed by treating the resin withPd(PPh₃)₄ (0.06 mmol, 69 mg, 3.0 equiv) in CHCl₃:AcOH:NEM (925:50:25)for 2 h. Washing is carried out with CHCl₃ (×6), a mixture of 5% sodiumdiethyldithiocarbamate trihydrate and 5% DIPEA in DMF (×2), and DMF(×10). The free amino group of the resin (ca. 0.02 mmol) is coupled withFmoc-Lys(Fmoc)-OH (0.06 mmol, 35 mg, 3.0 equiv.) via the TBTU activationprocedure, using TBTU (0.058 mmol, 19 mg, 2.88 equiv), and NEM (0.08mmol, 9 mg, 4.0 equiv). Following Fmoc-deprotection with 20% piperidinein DMF, first for 2 min, and then for 18 min, followed by washing withDMF (×6), the two newly liberated amino handles are coupled withFmoc-Lys(Fmoc)-OH (0.12 mmol, 71 mg, 3.0 equiv pr amino handle) via theTBTU activation procedure, using TBTU (0.115 mmol, 37 mg, 2.88 equiv.)and NEM (0.16 mmol, 18 mg, 4.0 equiv). Another round ofFmoc-deprotection with 20% piperidine in DMF, first for 2 min, and thenfor 18 min, followed by washing with DMF (×6), provided four aminohandles, which are coupled to the adhesion molecule AM-OH (0.24 mmol,534 mg, 3.0 equiv) via the TBTU activation procedure, using TBTU (0.23mmol, 73 mg, 2.88 equiv.) and NEM (0.32 mmol, 37 mg, 4.0 equiv). Theresin is washed with DMF (×6), and CH₂Cl₂ (×6), and lyophilizedovernight. Finally, the library synthesis is finished by treating theresin with 10% TFA (aq) for 24 h, followed by washing with water (×6),DMF (×6), and CH₂Cl₂ (×6). The resin is lyophilized overnight, and keptin the freezer (−18° C.).

Example 6b Library of Multi-Heterocyclic Peptidomimetics for GPCRReceptors (Library 6b).

This library is for example useful for identification of compoundsmodulating a cellular response mediated through a G-protein coupledreceptor.

Library Design and Synthesis

All Pictet-Spengler reaction methodology has been developed and testedon the synthesis resin PEGA₈₀₀,¹ wherefore the analogous library resinPEGA₁₉₀₀ is chosen for the library synthesis. In order to screen foractive compounds, the library is prepared following a“one-bead-two-compounds” strategy. This is accomplished by treating theamino-functionalized resin with a mixture of Fmoc-Gly-OH:Alloc-Gly-OH(1:1) activated by the TBTU procedure² to provide orthogonal reactionsites for (a) split-and-mix library synthesis (via the Fmoc handle); and(b) attachment of an adhesion molecule (AM) (via the Alloc handle). Thelibrary synthesis of Pictet-Spengler reaction precursors 3 is carriedout according to standard Fmoc amino acid coupling protocols forsolid-phase peptide synthesis (FIG. 6 b). Due to the requirement ofacidic reaction conditions for the Pictet-Spengler reaction step (q),the base labile HMBA (hydroxymethylbenzoic acid) linker is employed.Prior to attachment of HMBA to H₂N-Gly-PEGA₁₉₀₀ via the TBTU activationprocedure, the Fmoc protecting group is removed by standard piperidinetreatment. The HMBA linker provides a convenient cleavage site forquantitative release from the solid support via basic hydrolysis.Cleavage of product from a single bead is routinely achieved by treatingthe bead with 0.1 M NaOH (aq) overnight, thus providing amounts ofmaterial sufficient for structure elucidation via QTOF ES-MSMS analysis.The hydroxy handle of the linker is esterified by treatment withMSNT-activated Fmoc-Gly-OH³ thus placing glycine as the first amino acidresidue of the peptidomimetic sequence. Subsequent analogoussplit-and-mix synthesis and 4 cycles of Fmoc deprotection/TBTU-mediatedcouplings of 20 Fmoc amino acids as the first amino acid residue(Fmoc-AA₁-OH), 20 Fmoc amino acids as the second amino acid residue(Fmoc-AA₂-OH), 15 Fmoc amino acids incorporating the reactive aromaticside-chain (Fmoc-AA₃-OH), and 6 masked aldehyde building blocks(R⁴-MABB-OH) (table 5b), prepared as previously reported,^(4,5) affordthe Pictet-Spengler reaction precursor 3. The Alloc protecting group of3 is removed with Pd(PPh₃)₄, and subsequent TBTU coupling ofFmoc-Lys(Fmoc)-OH/Fmoc deprotection provided the amino handles forattachment of the adhesion molecule AM, which is accomplished via theTBTU activation procedure. The adhesion molecule is synthesized viastandard solid-phase peptide synthesis, and purified by preparative HPLCprior to attachment to resin. To finalize the library synthesis, theresin 4 is treated with 10% TFA (aq) to facilitate the intramolecularN-acyliminium Pictet-Spengler reaction andTFA:CH₂Cl₂:H₂O:MeSPh:(CH₂SH)₂:TIPS (66.5:20:5:5:2.5:1) to removeresidual protecting groups in the side-chains of AA₁ (R¹) and AA₂ (R²).As a consequence of the structurally diverse aromatic heterocyclesundergoing the intramolecular N-acyliminium Pictet-Spengler reaction,the library is graphically represented by the six sublibraries (Ib-VIb)below (FIG. 6 b). Theoretically, the library is composed by 38400different compounds (118800 different compounds when all stereoisomersare counted).

An overview of the synthesis of a combinatorial library via theintramolecular N-acyliminium Pictet-Spengler reaction^(a,b,c) is givenin FIG. 6 b. The amino acids and building blocks used for the librarysynthesis are indicated in table 5b.

Reagents and conditions: (a) Fmoc-Gly-OH:Alloc-Gly-OH (1:1), TBTU, NEM,DMF; (b) 20% piperidine (DMF); (c) HMBA, TBTU, NEM, DMF; (d)Fmoc-Gly-OH, MSNT, MeIm, CH₂Cl₂; (e) 20% piperidine (DMF); (f)Fmoc-AA₁OH, TBTU, NEM, DMF; (g) 20% piperidine (DMF); (h) Fmoc-AA₂-OH,TBTU, NEM, DMF; (i) 20% piperidine (DMF); (j) Fmoc-AA₃-OH, TBTU, NEM,DMF; (k) 20% piperidine (DMF); (l) R⁴-MABB-OH, TBTU, NEM, DMF; (m)Pd(PPh₃)₄ (CHCl₃:AcOH:NEM (925:50:25); (n) Fmoc-Lys(Fmoc)-OH, TBTU, NEM,DMF; (o) 20% piperidine (DMF); (p) AM-OH, TBTU, NEM, DMF, 20 h; (q) 10%TFA (aq); (r) TFA:CH₂Cl₂:H₂O:MeSPh:(CH₂SH)₂:TIPS(66.5:20:5:5:2.5:1).^(a) Sublibrary Ib consists of 26400 differentcompounds (92400 when all stereoisomers are counted).^(b) SublibrariesIIb, IIIb, IVb, and Vb each consists of 2400 different compounds (4400when all stereoisomers are counted). Sublibrary VIb consists of 2400different compounds (8800 when all stereoisomers are counted).

TABLE 5b Amino acids and building blocks for combinatorial librarysynthesis

AA₁ AA₂ AA₃ (Sublibrary structure) R⁴ His(Boc) His(Boc) Trp (Ib) HAsp(t-Bu) Asp(t-Bu) D/L-(5-Br)Trp (Ib) Me Arg(Pmc) Arg(Pmc) L-(5-OH)Trp(Ib) i-Bu Phe Phe D/L-(5-MeO)Trp (Ib) Bn Ala Ala D/L-(4-Me)Trp (Ib) PhCys(Trt) Cys(Trt) D/L-(5-Me)Trp (Ib) CH₂OH Gly Gly D/L-(6-Me)Trp (Ib)Gln(Trt) Gln(Trt) D/L-(5-BnO)Trp (Ib) Glu(t-Bu) Glu(t-Bu) D/L-(5-F)Trp(Ib) Lys(Boc) Lys(Boc) D/L-(6-F)Trp (Ib) Leu Leu L-(2-Thi)Ala (IIb) MetMet L-(3-Thi)Ala (IIIb) Asn(Trt) Asn(Trt) L-(2-Fur)Ala (IVb) Ser(t-Bu)Ser(t-Bu) L-(3-BzThi)Ala (Vb) Tyr(t-Bu) Tyr(t-Bu) D/L-(7-Aza)Trp (VIb)Thr(t-Bu) Thr(t-Bu) Ile Ile Trp(Boc) Trp(Boc) Pro Pro Val Val

General Methods. All solvents are of HPLC quality and stored overmolecular sieves. Solid-phase organic combinatorial chemistry isroutinely carried out using a 20-well peptide synthesizer equipped withsintered teflon filters (50 μm pores), teflon tubing, and valves, whichallow suction to be applied below the wells. For all reactions on solidsupport, PEGA₁₉₀₀ resin (0.24 mmol/g, VersaMatrix A/S) is used. Prior touse, the resin is washed with methanol (×6), DMF (×6), and CH₂Cl₂ (×6).All commercially available reagents are used as received without furtherpurification.

Analysis of all solid-phase reactions is performed after cleaving theproducts as their free acids from the resin. A single bead is treatedwith 0.1 M aqueous NaOH (10 μL) in a 0.5 mL Eppendorf tube overnight,then diluted with CH₃CN (20 μL), before filtering the solution, therebyproviding a sample for ES MSMS analysis on a MicroMass QTOF GlobalUltima mass spectrometer (mobile phase 50% CH₃CN (aq), 0.1 μL/min)employing a linear ramping of the collision energy. Spectra (FIG. 7) areanalyzed by generating the exact mass differences between fragment ionsand tabulated (FIG. 8) to provide the fragmentation pathway (FIG. 9) andtherefore structure of the compound released from the single bead.

Solid-phase synthesis of combinatorial library (6b). Attachment ofFmoc-Gly-OH/Alloc-Gly-OH to the amino-functionalized PEGA₁₉₀₀ resin(0.24 mmol/g, 1.68 mmol, 7.00 g). The resin swelled in DMF is addedsolutions (i)+(ii) of TBTU-activated N-protected glycines; (i)Fmoc-Gly-OH (1.5 equiv., 2.52 mmol, 749 mg)+NEM (2.0 equiv., 3.36 mmol,426 μL)+TBTU (1.44 equiv., 2.42 mmol, 809 mg) in 5 mL DMF (activation inthe usual way); and (ii) Alloc-Gly-OH (1.5 eq, 2.52 mmol, 401 mg)+NEM(2.0 eq, 3.36 mmol, 426 μL)+TBTU (1.44 eq, 2.42 mmol, 809 mg) in 5 mLDMF (activation in the usual way). Both solutions are simultaneouslyadded to the resin in 100×50 μL portions with vigorous shaking,maintaining the rate at 1 addition from each solution pr. minute. Afteraddition of solutions (i) and (ii), the reaction mixture is furthershaken for 30 min, followed by washing with DMF (×6), and CH₂Cl₂ (×6) ina syringe fitted with a Teflon filter. Completion of the reaction ismonitored using the Kaiser test. Prior to attachment of the HMBA linkervia the procedure above, Fmoc-deprotection is accomplished with 20%piperidine in DMF, first for 2 min, and then for 18 min, followed bywashing with DMF (×6). Coupling of the first amino acid (Fmoc-Gly-OH) tothe HMBA derivatized resin is accomplished by treating the freshlylyophilized resin (0.84 mmol) with a mixture of the Fmoc-Gly-OH (4 eq,3.4 mmol, 999 mg), MeIm (8 eq, 6.8 mmol, 533 μL), and MSNT (4 eq, 3.4mmol, 996 mg) in dry CH₂Cl₂ (30 mL).³ The coupling is carried out for 2h, then the resin is washed with dry DMF (×1), and dry CH₂Cl₂ (×1),before repeating the MSNT coupling of Fmoc-Gly-OH once. The resin iswashed with DMF (×6) and CH₂Cl₂ (×6) prior to lyophilization for removalof all solvent traces. A batch of resin (1.00 g) is subjected tosplit-and-mix peptide syntheses with Fmoc-AA₁-OH, Fmoc-AA₂-OH,Fmoc-AA₃-OH, and R⁴-MABB-OH, respectively, following the couplingprocedure described above for the attachment of Fmoc-Gly-OH (via TBTUand NEM in DMF).² The usual washing protocol follows each coupling anddeprotection step, and all couplings are checked via the Kaiser test.The Alloc group of 3 is removed by treating the resin batch twice withPd(PPh₃)₄ (3.0 equiv., 0.36 mmol, 416 mg) in CHCl₃:AcOH:NEM (925:50:25)for 3 h. Washing was carried out with CHCl₃ (×6) and DMF (×10). The freeamino group of the resin (0.12 mmol) was coupled with Fmoc-Lys(Fmoc)-OH(3.0 equiv., 0.36 mmol, 210 mg) via the TBTU activation procedure, usingTBTU (2.88 equiv., 0.348 mmol, 114 mg) and NEM (4.0 equiv., 0.48 mmol,54 mg). Following Fmoc-deprotection with 20% piperidine in DMF, firstfor 2 min, and then for 18 min, followed by washing with DMF (×6), newlyliberated amino handle is coupled to the adhesion molecule AM-OH (1.5equiv., 0.36 mmol, 801 mg) via the TBTU activation procedure, using TBTU(2.88 equiv., 0.691 mmol, 222 mg) and NEM (4.0 equiv., 0.96 mmol, 122μL). The resin was washed with DMF (×6), and CH₂Cl₂ (×6), andlyophilized overnight. The library synthesis was finished by firsttreating the resin with 10% TFA (aq) for 24 h, followed by washing withwater (×6), DMF (×6), and CH₂Cl₂ (×6), and finally withTFA:CH₂Cl₂:H₂O:MeSPh:(CH₂SH)₂:TIPS (66.5:20:5:5:2.5:1) for 5 h, beforewashing with CH₂Cl₂ (×6), DMF (×6), water (×6), DMF (×6), and CH₂Cl₂(×6). The resin was lyophilized overnight, and stored in the freezer(−18° C.).

REFERENCES

-   (1) Meldal, M. Tetrahedron Lett. 1993, 33, 3077-3080.-   (2) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D.    Tetrahedron Lett. 1989, 30, 1927-1930.-   (3) Blankemeyer-Menge, B.; Nimtz, M.; Frank, R. Tetrahedron Lett.    1990, 31, 1701-1704.-   (4) Groth, T.; Meldal, M. J. Comb. Chem. 2001, 3, 34-44.-   (5) Nielsen, T. E.; Meldal, M. J. Org. Chem. 2004, 69, 3765-3773.

Example 7

Gs Coupled Receptor (MC4R): Agonist Assay (Cre-GFPreporter AssayDetected with a Fluorescence Activated Bead Sorter)

Cre-GFP:

Cre-GFP is commercially available from clontech (pCre-d2eGFP) The vectorcontains three copies of Cre-binding sequence fused to a TATA-likepromoter. The vector is holding a neomycin resistance gene. A map of thevector is shown in FIG. 3.

MC4R:

PCR amplified MC4R encoding DNA is introduced into the gateway EntryVector (pENTR) by topoisomarase-mediated ligation. The DNA issubsequently recombined into Destination Vector pDEST12.2.(pDEST12.2MC4R)

Cell Line Establishment:

U2OS cells are transfected with pDEST1.2.2MC4R using standard procedurefor Fugene6 transfection. Cells are put under G418 selection for 4 weeksto obtain a cell line stably expressing MC4R.

The U2OS cell line stably expressing the human MC4R (melanocortin4receptor) is further transfected with Cre-GFP the day before culturingthem on PEGA beads displaying adhesion peptide and respectively 1)Negative control (PEGA beads with adhesion peptide, but no librarycompound), 2) Positive control (PEGA beads of example 2) and 3) Librarycompounds. The three cultures are handled separately in each theirculture flask.

Bead/Cell Preparation:

Cells are trypsinized and mixed with the PEGA beads in growth medium(DMEM containing 10% FCS, in the proportion 4000 cells/bead and app. 50ml growth medium/5000 beads

1) Positive control: 50 ml Growth medium+5000 positive controlbeads+2×10E7 cells.2) Negative control: 50 ml Growth medium+5000 negative controlbeads+2×10E7 cells.3) Screening library (eg. 100,000 compounds): 1000 ml Growthmedium+100,000 library beads+4×10E8 cells.

The three culture flasks are placed on a Magnetic stirring platform(Techne) designed for cell culture in suspension and incubated at 37°,5% CO2 for 16-24 hrs using spinning interval 30 rpm, 3 min stirring, 10min pause. Beads, now covered with cells, are allowed to sediment for 10min (no centrifugation needed) and the growth medium is removed using a50 ml pipette. 10 ml 99% EtOH per 5000 beads is added, mixed gently andleft for 15 min. Beads are washed w. 10 ml PBS/5000 beads ×3 by allowingsedimentation for 10 min between each wash. Cells are now preserved andfixed to the beads

Bead Sorting:

A Fluorescence Activated Bead Sorter (FABS) equipped with a multilineArgon laser 488 nm excitation line and 500-650 nm emission filter andsorting capability into 96 well plate is used to identify and isolatepositive hit beads.

The FABS is calibrated to identify and isolate positive hit beads(increased GFP fluorescence) by determining the dynamic range of theassay using positive control beads prepared as described in Example 2 asSmax (maximum response) and negative control beads comprising only celladhesion peptide as 5 min (minimum response). A cut off at 30% responsecompared to negative control beads is set as threshold for a positivehit bead.

Positive hits are separated into each their well of a 96 well plate andare hereafter ready for compound elucidation, re-synthesis and re-testas well as test for effects in other assays.

This assay may also be performed using HEK cells essentially asdescribed herein below in Example 7a, except that the HEK cells shouldbe transfected with the Cre-GFP and pDEST1.2.2MC4R constructs. Positiveresin beads may also preferably be selected using a fluorescencemicroscope, as described in Example 7a.

Example 7a Gs Coupled Receptor (MC4R): Agonist Assay (MC4R-GFPInternalization: Microscopy) Construction of MC4R-GFP:

996 bp of MC4R ORF sequence without stop codon is inserted into pGFP2-N1vector (Biosignal Packard Cat. # 6310013-001) with cloning sitesEcoRI/BamHI.

Cell Line Establishment:

Hek293 cells are transfected with MC4R-GFP using standard procedure forFugene6 transfection. Cells are put under zeocin selection for 4 weeksto obtain a cell line stably expressing the MC4R-GFP.

Cell/Bead Preparation:

Cells were cultured on respectively 1) Negative control beads (preparedas described in example 1), 2) Positive control beads (prepared asdescribed in example 2; Ac-His-(D)phe-Arg-Trp-Gly-PEGA₁₉₀₀) and Librarybeads (prepared as described in example 6b). Each batch of beads washandled separately.

Cells were trypsinized and mixed with Negative control beads/Positivecontrol beads/Library beads in growth medium (Hams F12 containing 5%FCS)

-   -   Add 500 beads in 500 ul Hams to a 14 ml Nunc tube    -   Add 2500 ul cell suspension 1×10E6/ml Hams w. 5% FCS    -   Leave tube vertically in incubator (37 degrees, 5% CO2) for        16-24 hrs—rock tube gently every 15 min for the first hour    -   Remove medium. Wash loose cells away by gently adding and        removing 4 ml Hams ×2 (Turn the tube upside down and back        again—as soon as beads have sedimented suck away medium)    -   Add 2 ml Hams w. FCS 5%    -   Incubate o/n at 37 degrees, 5% CO2    -   Decant beads to a 1 well Lab-Tek Chambered Coverglass (#155361)

Hit Identification and Isolation

The LabTek 1 well chambered coverglass was placed on a Zeiss Axiovert200 fluorescence microscope equipped with filters optimal for GFPfluorescence. The microscope was further more equipped with amicromanipulator (Eppendorf Transferman NK2)) capable of picking outsingle beads. Using 40× objective chambers were scanned for positive hitbeads, which appeared as cells with green dots located in the cytoplasmain contrast to negative beads where GFP is located in the plasmamembrane of the cells. Positive and negative control beads were used toset cut off for positive hit beads. Such hit beads were picked out usingthe micromanipulator. MC4R-GFP internalization was quantified and theresults are shown in FIG. 11.

Example 8 Gs Coupled Receptor (MC4R): Agonist Assay (Multiplexed Cre-YFPReporter and MC4R-GFPinternalization: FABS and Microscopy)

Construction of pCRE-d2EYFP:

A 732 bp of EYFP fragment from pd2EYFP-1 (Clontech Cat. #6912-1) isligated to a 3.5 kb fragment from pCRE-d2EGFP (Clontech Cat #6034-1).Both fragments are excised from the two vectors by a common restrictionenzyme digestion.

Construction of MC4R-GFP:

996 bp of MC4R ORF sequence without stop codon is inserted into pGFP2-N1vector (Biosignal Packard Cat. # 6310013-001) with cloning sitesEcoRI/BamHI.

Cell Line Establishment:

U2OS cells are transfected with MC4R-GFP using standard procedure forFugene6 transfection. Cells are put under zeocin selection for 4 weeksto obtain a cell line stably expressing the MC4R-GFP.

The U2OS cell line stably expressing the human MC4R-GFP (melanocortin4receptor-GFP) is further transfected with Cre-YFP the day beforeculturing them on PEGA beads displaying adhesion peptide andrespectively 1) Negative control (PEGA beads with adhesion peptide, butno library compound), 2) Positive control (PEGA beads of example 2) and3) Library compounds. The three cultures are handled separately in eachtheir culture flask.

Bead/Cell Preparation:

Cells are trypsinized and mixed with beads in growth medium (DMEMcontaining 10% FCS, in the proportion 4000 cells/bead and app. 50 mlgrowth medium/5000 beads.

1) Positive control: 50 ml Growth medium+5000 positive controlbeads+2×10E7 cells.2) Negative control: 50 ml Growth medium+5000 negative controlbeads+2×10E7 cells.3) Screening library (eg. 100,000 compounds): 1000 ml Growthmedium+100,000 library beads+4×10E8 cells

The three culture flasks are placed on a Magnetic stirring platform(Techne) designed for cell culture in suspension and incubated at 37°,5% CO2 for 16-24 hrs using spinning interval 30 rpm, 3 min stirring, 10min pause.

Beads, now covered with cells, are allowed to sediment for 10 min (nocentrifugation needed) and the growth medium is removed using a 50 mlpipette. 10 ml 99% EtOH per 5000 beads is added, mixed gently and leftfor 15 min. Beads are washed w. 10 ml PBS/5000 beads ×3 by allowingsedimentation for 10 min between each wash. Cells are now preserved andfixed to the beads

Bead Sorting for Cre-YFP Response:

A Fluorescence Activated Bead Sorter (FABS) equipped with 514 nmexcitation laser line and 528-572 nm emission filter is used to identifyand isolate positive hit beads.

The FABS is calibrated to identify and isolate positive hit beads(increased YFP fluorescence) by determining the dynamic range of theassay using positive control beads as Smax (maximum response) andnegative control beads as 5 min (minimum response). A cut off at 30%response compared to negative control beads is set as threshold for apositive hit bead.

Positive hit beads are isolated into a 1 well Nunc chamber and arehereafter ready to test for receptor internalisation.

MC4R-GFPinternalisation: Microscope Analysis

The Nunc chamber with positive Cre-YFP hits is placed on an imagingmicroscope (Zeiss Axiovert 200M) equipped with filters allowingseparation of YFP and GFP. Further more the microscope is equipped witha micromanipulator (Eppendorf Transferman NK2)) capable of picking outsingle beads. Using 20× objective the chamber is scanned for positiveMC4R-GFPinternalisation (appear as intracellular spots as compared tomembrane distribution in non positive MC4R-GFP internalisation) and suchhit beads are picked out using the micromanipulator for compoundstructure elucidation.

A multiplexed screening like this is expected to give very low rate offalse positive hits since hits picked out for structure elucidation aregiving rise to both specific receptor activation (internalisation ofreceptor) as well as a functional response (activation of transcriptionof Cre-YFP).

Example 9 Gs Coupled Receptor (MC4R): Agonist Assay (Cre-YFP Reporterand HA-MC4R Internalization: FABS and Microscopy)

Construction of pCRE-d2EYFP:

A 732 bp of EYFP fragment from pd2EYFP-1 (Clontech Cat. #6912-1) isligated to a 3.5 kb fragment from pCRE-d2EGFP (Clontech Cat #6034-1).Both fragments are excised from the two vectors by a common restrictionenzyme digestion.

Construction of HA-MC4R:

999 bp of MC4R ORF sequence is first inserted into pCMV-HA vector(Clontech Cat#6003-1) with cloning sites EcoRI/XhoI, then the fusionfragment of HA-MC4R is further cloned into pcDNA3.1/Zeo (InvitrogenCat.#V86520) with the cloning sites HindIII/XhoI.

Cell Line Establishment:

U2OS cells are transfected with HA-MC4R using standard procedure forFugene6 transfection. Cells are put under zeocin selection for 4 weeksto obtain a cell line stably expressing the HA-MC4R.

The U2OS cell line stably expressing the human HA-MC4R (melanocortin4receptor-GFP) is further transfected with Cre-YFP the day beforeculturing them on PEGA beads displaying adhesion peptide andrespectively 1) Negative control (PEGA beads with adhesion peptide, butno library compound), 2) Positive control (PEGA beads of example 2) and3) Library compounds. The three cultures are handled separately in eachtheir culture flask.

Bead/Cell Preparation:

Cells are trypsinized and mixed with beads in growth medium (DMEMcontaining 10% FCS, in the proportion 4000 cells/bead and app. 50 mlgrowth medium/5000 beads.

1) Positive control: 50 ml Growth medium+5000 positive controlbeads+2×10E7 cells.2) Negative control: 50 ml Growth medium+5000 negative controlbeads+2×10E7 cells.3) Screening library (eg. 100,000 compounds): 1000 ml Growthmedium+100,000 library beads+4×10E8 cells

The three culture flasks are placed on a Magnetic stirring platform(Techne) designed for cell culture in suspension and incubated at 37°,5% CO2 for 16-24 hrs using spinning interval 30 rpm, 3 min stirring, 10min pause.

Beads, now covered with cells, are allowed to sediment for 10 min (nocentrifugation needed) and the growth medium is removed using a 50 mlpipette. 10 ml 99% EtOH per 5000 beads is added, mixed gently and leftfor 15 min. Beads are washed w. 10 ml PBS/5000 beads ×3 by allowingsedimentation for 10 min between each wash. Cells are now preserved andfixed to the beads

Bead Sorting for Cre-YFP Response:

A Fluorescence Activated Bead Sorter (FABS) equipped with a multilineArgon laser adjusted to the 514 nm excitation line and 528-572 nmemission filter is used to identify and isolate positive hit beads.

The FABS is calibrated to identify and isolate positive hit beads(increased YFP fluorescence) by determining the dynamic range of theassay using positive control beads as Smax (maximum response) andnegative control beads as 5 min (minimum response). A cut off at 30%response compared to negative control beads is set as threshold for apositive hit bead.

Positive hits are isolated into a 1 well Nunc chamber and are hereafterready to test for receptor internalisation.

HA-MC4R Internalisation: Microscope Analysis

Beads isolated as positive hits in Cre-YFP transcription reporter assayby FABS are treated with Triton-x to permeabilize cells followed byincubation with HA-tag poly-clonal antibody followed by staining withappropriate TRITC conjugated secondary antibody. A Nunc 1 well chamberholding the labelled beads are placed on an imaging microscope (ZeissAxiovert 200M) equipped with filters allowing separation of YFP andTRITC. Further more the microscope is equipped with a micromanipulator(Eppendorf Transferman NK2)) capable of picking out single beads. Using20× objective the chamber is scanned for positive HA-internalisation(appear as intracellular spots as compared to membrane distribution innon positive HA-MC4Rinternalisation and such hit beads are picked outfor compound structure elucidation.

A multiplexed screening like this is expected to give very low rate offalse positive hits since hits picked out for structure elucidation isgiving rise to both specific receptor activation (observed asinternalisation of receptor) as well as a functional response (observedas transcription of Cre-YFP construct).

Example 10 Gs Coupled Receptor (MC4R): Agonist Assay (Cre-YFP Reporterand HA-MC4R Internalization: FABS+FABS)

Construction of pCRE-d2EYFP:

A 732 bp of EYFP fragment from pd2EYFP-1 (Clontech Cat.#6912-1) isligated to a 3.5 kb fragment from pCRE-d2EGFP (Clontech Cat #6034-1).Both fragments are excised from the two vectors by a common restrictionenzyme digestion.

Construction of HA-MC4R:

999 bp of MC4R ORF sequence is first inserted into pCMV-HA vector(Clontech Cat#6003-1) with cloning sites EcoRI/XhoI, then the fusionfragment of HA-MC4R is further cloned into pcDNA3.1/Zeo (InvitrigenCat.#V86520) with the cloning sites HindIII/XhoI.

Cell Line Establishment:

U2OS cells are transfected with HA-MC4R using standard procedure forFugene6 transfection. Cells are put under zeocin selection for 4 weeksto obtain a cell line stably expressing the HA-MC4R.

The U2OS cell line stably expressing the human HA-MC4R (HA-melanocortin4receptor) is further transfected with Cre-YFP the day before culturingthem on PEGA beads displaying adhesion peptide and respectively 1)Negative control (PEGA beads with adhesion peptide, but no librarycompound), 2) Positive control (PEGA beads of example 2) and 3) Librarycompounds. The three cultures are handled separately in each theirculture flask.

Bead/Cell Preparation:

Cells are trypsinized and mixed with beads in growth medium (DMEMcontaining 10% FCS, in the proportion 4000 cells/bead and app. 50 mlgrowth medium/5000 beads.

1) Positive control: 50 ml Growth medium+5000 positive controlbeads+2×10E7 cells.2) Negative control: 50 ml Growth medium+5000 negative controlbeads+2×10E7 cells.3) Screening library (eg. 100,000 compounds): 1000 ml Growthmedium+100,000 library beads+4×10E8 cells

The three culture flasks are placed on a Magnetic stirring platform(Techne) designed for cell culture in suspension and incubated at 37°,5% CO2 for 16-24 hrs using spinning interval 30 rpm, 3 min stirring, 10min pause. Beads, now covered with cells, are allowed to sediment for 10min (no centrifugation needed) and the growth medium is removed using a50 ml pipette. 10 ml 99% EtOH per 5000 beads is added, mixed gently andleft for 15 min. Beads are washed w. 10 ml PBS/5000 beads ×3 by allowingsedimentation for 10 min between each wash. Cells are now preserved andfixed to the beads

Bead Sorting for Cre-YFP Response:

A Fluorescence Activated Bead Sorter (FABS) equipped with 514 nm laserexcitation line and 528-572 nm emission filter is used to identify andisolate positive hit beads.

The FABS is calibrated to identify and isolate positive hit beads(increased YFP fluorescence) by determining the dynamic range of theassay using positive control beads as Smax (maximum response) andnegative control beads as 5 min (minimum response). A cut off at 30%response compared to negative control beads is set as threshold for apositive hit bead.

Positive hits are isolated into a 10 ml tube and are hereafter ready totest for receptor internalisation.

HA-MC4R Internalisation: FABS Analysis

A Fluorescence Activated Bead Sorter (FABS) equipped with 568 nm laserline excitation and 584-640 nm emission filter is used to identify andisolate positive hit beads.

Beads isolated as positive hits in Cre-YFP transcription reporter assayby FABS as well as positive and negative control beads are incubatedwith HA-tag polyclonal antibody followed by staining with appropriateTRITC conjugated secondary antibody.

The FABS is calibrated to identify and isolate positive hit beads bydetermining the dynamic range of the assay using positive control beadsas Smax (maximum response) and negative control beads as 5 min (minimumresponse). A cut off at 30% response compared to negative control beadsis set as threshold for a positive hit bead. Positive hits are givingless TRITC fluorescence than negative hits caused by receptorinternalization resulting in inability of the TRITC conjugated sec.antibody to reach the HA-tag (no permeabilization of the plasmamembrane).

Positive hits are separated into each well of a 96 well plate and arehereafter ready for compound elucidation, re-synthesis and re-test aswell as test for effects in other assays.

A multiplexed screening like this is expected to give very low rate offalse positive hits since hits picked out for structure elucidation isgiving rise to both specific receptor activation as well as a functionalresponse

Example 11

Gs coupled Receptor (β2AR): Antagonist Assay (Cre-Reporter)

Cre-GFP:

Cre-GFP (c-AMP Response Element-Green Fluorescent Protein) commerciallyavailable from clontech (pCre-d2eGFP) The vector contains three copiesof Cre-binding sequence fused to a TATA-like promoter. The vector isholding a neomycin resistance gene. A map of the vector is shown in FIG.3. β2 adrenergic receptor (β2AR):

A 1776 bp cDNA fragment containing β2AR ORF sequence is PCR-amplifiedfrom human kidney and fetal brain cDNA libraries (Clontech Cat#639305,6393029) using primers designed from β2AR mRNA sequence (accession #NM_(—)000024), and cloned into pCR-XL-TOPO vector (invitrogen). A 1274bp of β2AR gene containing a kozak sequence and a stop codon is furthercloned into pcDNA3.1/zeo(+) vector (invitrogen) with the restrictionsites HindIII/XhoI. The β2AR gene is sequencing confirmed.

Cell Line Establishment:

U2OS cells are transfected with β2AR using standard procedure forFugene6 transfection. Cells are put under zeocin selection for 4 weeksto obtain a cell line stably expressing the β2AR.

The U2OS cell line stably expressing the human β2AR is furthertransfected with Cre-GFP the day before culturing them on PEGA beadsdisplaying adhesion peptide and respectively 1) Negative control (PEGAbeads with adhesion peptide, but no library compound), 2) Positivecontrol (PEGA beads displaying adhesion peptide and isoproterenol) and3) Library compounds. The three cultures are handled separately in eachtheir culture flask.

Bead/Cell Preparation:

Cells are trypsinized and mixed with beads in growth medium (DMEMcontaining 10% FCS), added Isoproterenol 10 uM, in the proportion 4000cells/bead and app. 50 ml growth medium w. isoproterenol 10 uM/5000beads.

1) Positive control: 50 ml Growth medium w. proterenol 10 uM+5000positive control beads+2×10E7 cells.2) Negative control: 50 ml Growth medium w. proterenol 10 uM+5000Negative control beads+2×10E7 cells.3) Screening library (eg. 100,000 compounds): 1000 ml Growth medium w.proterenol 10 uM+100,000 library beads+4×10E8 cells

The three culture flasks are placed on a Magnetic stirring platform(Techne) designed for cell culture in suspension and incubated at 37°,5% CO2 for 16-24 hrs using spinning interval 30 rpm, 3 min stirring, 10min pause.

Beads, now covered with cells, are allowed to sediment for 10 min (nocentrifugation needed) and the growth medium is removed using a 50 mlpipette. 10 ml 99% EtOH per 5000 beads is added, mixed gently and leftfor 15 min. Beads are washed w. 10 ml PBS/5000 beads ×3 by allowingsedimentation for 10 min between each wash. Cells are now preserved andfixed to the beads

Bead Sorting:

A Fluorescence Activated Bead Sorter (FABS) equipped with 488 nm laserexcitation line and 500-550 nm emission filter is used to identify andisolate positive hit beads (=inhibition of isoproterenol induced Cre-GFPtranscription (=decreased fluorescence compared to negative control).

The FABS is calibrated to identify and isolate positive hit beads bydetermining the dynamic range of the assay using positive control beadsas Smax (maximum inhibition=minimal fluorescence) and negative controlbeads as 5 min (minimum inhibition=maximal fluorescence). A cut off at30% inhibition compared to negative control beads is set as thresholdfor a positive hit bead.

Positive hits are separated into each their well of a 96 well plate andare hereafter ready for compound elucidation, re-synthesis and re-testas well as test for effects in other assays.

Example 12 Gi Coupled Receptor (CCR5): Agonist Assay (Cre-GFP Reporter)Cre-GFP:

Cre-GFP is commercially available from clontech (pCre-d2eGFP) The vectorcontains three copies of Cre-binding sequence fused to a TATA-likepromoter. The vector is holding a neomycin resistance gene. A map of thevector is shown in FIG. 3. C—C Chemokine Receptor5 (CCR5):

Accession no. AAB57793

U2OS cells are transfected with CCR5 using standard procedure forFugene6 transfection. Cells are put under zeocin selection for 4 weeksto obtain a cell line stably expressing the CCR5.

The U2OS cell line stably expressing the human CCR5 is furthertransfected with Cre-GFP the day before culturing them on PEGA beadsdisplaying adhesion peptide and respectively 1) Negative control (PEGAbeads with adhesion peptide, but no library compound), 2) Positivecontrol (PEGA beads with adhesion peptide and RANTES) and 3) Librarycompounds. The three cultures are handled separately in each theirculture flask.

Bead/Cell Preparation:

Cells are trypsinized and mixed with beads in DMEM containing 10% FCS,10 uM forskolin and 500 uM IBMX, in the proportion 4000 cells/bead andapp. 50 ml DMEM/5000 beads.

1) Positive control: 50 ml DMEM+5000 positive control beads+2×10E7cells.2) Negative control: 50 ml DMEM+5000 negative control beads+2×10E7cells.3) Screening library (eg. 100,000 compounds): 1000 ml DMEM+100,000library beads+4×10E8 cells

The three culture flasks are placed on a Magnetic stirring platform(Techne) designed for cell culture in suspension and incubated at 37°,5% CO2 for 16-24 hrs using spinning interval 30 rpm, 3 min stirring, 10min pause.

Beads, now covered with cells, are allowed to sediment for 10 min (nocentrifugation needed) and the growth medium is removed using a 50 mlpipette. 10 ml 99% EtOH per 5000 beads is added, mixed gently and leftfor 15 min. Beads are washed w. 10 ml PBS/5000 beads ×3 by allowingsedimentation for 10 min between each wash. Cells are now preserved andfixed to the beads

Bead Sorting:

A Fluorescence Activated Bead Sorter (FABS) equipped with 488 nm laserexcitation line and 500-550 nm emission filter and sorting capabilityinto 96 well plate is used to identify and isolate positive hit beads.

The FABS is calibrated to identify and isolate positive hit beads(decreased GFP fluorescence compared to negative control) by determiningthe dynamic range of the assay using positive control beads (RANTES) asSmax (maximum response=minimal fluorescence) and negative control beadsas 5 min (minimum response=maximal fluorescence). A cut off at 30%response compared to negative control beads is set as threshold for apositive hit bead.

Positive hits are separated into each their well of a 96 well plate andare hereafter ready for compound elucidation, re-synthesis and re-testas well as test for effects in other assays.

Example 13 Gq Coupled Receptor (Muscarinic M1): Antagonist Assay (Ca++Mobilization Using Fluo-4) Ca++ Antagonist Assay:

This assay is designed to identify muscarinic M1 antagonist compounds.The read out is changes in intracellular Ca++ conc. detected using theFluo-4 probe from Molecular probes (see description elsewhere). Positivehits are compounds that inhibit Carbacol (muscarinic M1 agonist) inducedincrease in intracellular Ca++. U2OS cells are transfected withMuscarinic M1 receptor using standard procedure for Fugene6transfection. Cells are put under zeocin selection for 4 weeks to obtaina cell line stably expressing the Muscarinic M1 receptor.

U2OS cells expressing the Muscarinic M1 receptor are cultured on PEGAbeads displaying adhesion peptide and respectively 1) Negative control(Beads comprising only cell adhesion compound), 2) Positive control(beads comprising cell adhesion compound and Atropine) and 3) Librarycompounds. The three cultures are handled separately in each theirculture flask.

Bead/Cell Preparation:

Cells are trypsinized and mixed with beads in DMEM containing 10% FCS inthe proportion 4000 cells/bead and app. 50 ml growth medium/5000 beads.

1) Positive control: 50 ml Growth medium+5000 positive controlbeads+2×10E7 cells.2) Negative control: 50 ml Growth medium+5000 negative controlbeads+2×10E7 cells.3) Screening library (eg. 100,000 compounds): 1000 ml Growthmedium+100,000 library beads+4×10E8 cells

The three culture flasks are placed on a Magnetic stirring platform(Techne) designed for cell culture in suspension and incubated at 37°,5% CO2 for 16-24 hrs using spinning interval 30 rpm, 3 min stirring, 10min pause.

Measurement of changes in the cytoplasmic free calcium concentration[Ca²⁺]_(i) Beads, now covered with cells, are allowed to sediment for 10min (no centrifugation needed) and the growth medium is removed using a50 ml pipette. 10 ml Krebs Ringer buffer (KRW; KrebsRingerWollheim, pH7.4: NaCl 0.14 M, KCL 3.6 mM, NaH₂PO₄, H₂O 0.5 mM, MgSO₄, 7H₂O 0.5.mM,NaHCO₃, 2H₂O 1.5 mM, D-Glucose 6 mM, CaCl₂1.5 mM, HEPES 10 mM) added 1uM Fluo-4 (Molecular Probes F-14201)+0.02% Pluronic (Molecular ProbesF-127) per 5000 beads is added, mixed gently and cells/beads areincubated at 37° c. for 30 min. Beads are hereafter washed w. 10 mlKRW/5000 beads ×3 by allowing sedimentation for 10 min between eachwash. The Fluo-4 loaded cells are now ready for detection of changes in[Ca²⁺]_(i).

The fluorescence is monitored in either a Fluorescence Activated BeadSorter (FABS) (COPAS from Union Biometrica, US) that is equipped withmultiple laser excitation lines (476 nm, 483 nm, 488 nm, 496 nm, 514 nm,520 nm, 568 nm, 647 nm, 676 nm) or a fluorescence plate-reader(Polarstar Optima from BMG Labtech, Germany) that is equipped with aflash Xenon blitz lamp. Fluo4 fluorescence is detected in FABS byexciting with 488 nm and collecting the emitted light on to a PMTthrough a 530±30 nm emission filter, and on the plate-reader the cellsare excited through a 490±5 nm excitation filter and the emissioncollected through a 510±5 nm emission filter. For calculation of theexact [Ca²⁺]_(i), the fluorescence intensity is converted to [Ca²⁺]_(i);by using the equation [Ca²⁺]_(i)=K_(D)[F−F_(min))/(F_(max)−F)] where thedissociation constant K_(D) is 345 nM, F is fluorescence intensity,F_(min) is total fluorescence in the absence of Ca²⁺ and F_(max) istotal fluorescence when Fluo3 is saturated with Ca²⁺. To obtain F_(min)the cells are pre-incubated in a calcium low buffer (pH 7.4: NaCl 0.14M, KCL 3.6 mM, NaH₂PO₄, H₂O 0.5 mM, MgSO₄, 7H₂O 0.5.mM, NaHCO₃, 2H₂O 1.5mM, D-Glucose 6 mM, EGTA 1.5 mM, HEPES 10 mM) and is challenged with 1uM ionomycin immediately before the fluorescence detection. SimilarlyF_(max) is obtained by suspending the cells in a calcium saturatedbuffer (pH 7.4: NaCl 0.14 M, KCL 3.6 mM, NaH₂PO₄, H₂O 0.5 mM, MgSO₄,7H₂O 0.5.mM, NaHCO₃, 2H₂O 1.5 mM, D-Glucose 6 mM, CaCl₂1.5 mM, HEPES 10mM) and challenged with 1 uM ionomycin immediately before detection.

In several of our screening assay we do not use exact ion [Ca²⁺]_(i),but express the response of screening compounds as relative to controlcompounds (see below).

Bead Sorting for Fluo-4 Signal:

A Fluorescence Activated Bead Sorter (FABS) equipped with 488 nm laserexcitation line and 528-572 nm emission filter and injection capabilityis used to identify and isolate positive hit beads (=inhibition ofCarbachol induced Ca++ response=decreased fluorescence compared tonegative control).

The FABS is calibrated to identify and isolate positive hit beads bydetermining the dynamic range of the assay using positive control beadsas Smax (maximum inhibition=minimal fluorescence) and negative controlbeads as 5 min (minimum inhibition=maximal fluorescence). Carbacol 1 uMis injected into the flow steam resulting in an increase in fluorescencefor negative control beads and an unchanged or minor increase influorescence for positive control beads. A cut off at 30% inhibitioncompared to negative control beads is set as threshold for a positivehit bead. Positive hit beads may preferably be identified using a platereader essentially as described in Example 13a herein below.

Positive hits are separated into each their well of a 96 well plate andare hereafter ready for compound elucidation, re-synthesis and re-testas well as test for effects in other assays.

Example 13a

Gq Coupled Receptor (Muscarinic M1): Antagonist Assay (Ca++ Mobilizationusing Fluo-4)

Ca++Antagonist Assay:

This assay is designed to identify muscarinic M1 antagonistic compounds.The readout is changes in intracellular Ca++ conc. detected using theFluo-4 probe from Molecular probes. Positive hits are compounds thatinhibit Carbacol (muscarinic M1 agonist) induced increase inintracellular Ca++.

For calculation of the exact [Ca²⁺]_(i) the fluorescence intensity isconverted to [Ca²⁺]_(i), by using the equation[Ca²⁺]_(i)=K_(D)[(F−F_(min))/(F_(max)−F)] where the dissociationconstant K_(D) is 345 nM, F is fluorescence intensity, F_(min) is totalfluorescence in the absence of Ca²⁺ and F_(max) is total fluorescencewhen Fluo4 is saturated with Ca²⁺. To obtain F_(min) the cells arepre-incubated in a calcium low buffer (pH 7.4: NaCl 0.14 M, KCL 3.6 mM,NaH₂PO₄, H₂O 0.5 mM, MgSO₄, 7H₂O 0.5.mM, NaHCO₃, 2H₂O 1.5 mM, D-Glucose6 mM, EGTA 1.5 mM, HEPES 10 mM, probenecid 2 mM) and is challenged with1 uM ionomycin immediately before the fluorescence detection. SimilarlyF_(max) is obtained by suspending the cells in a calcium saturatedbuffer (pH 7.4: NaCl 0.14 M, KCL 3.6 mM, NaH₂PO₄, H₂O 0.5 mM, MgSO₄,7H₂O 0.5.mM, NaHCO₃, 2H₂O 1.5 mM, D-Glucose 6 mM, CaCl₂1.5 mM, HEPES 10mM, probenecid 2 mM) and challenged with 1 uM ionomycin immediatelybefore detection.

In general it is not required to use exact ion [Ca²⁺]_(i). Rather, theresponse of screening compounds may be expressed as relative to controlcompounds (see below).

Cell Line Establishment:

BHK cells are transfected with the muscarinic M1 receptor using standardprocedure for Fugene6 transfection. Cells are put under G418 selectionfor 4 weeks to obtain a cell line stably expressing the muscarinic M1receptor.

Cell/Bead Preparation:

BHK cells expressing the Muscarinic M1 receptor are cultured on Negativecontrol beads (prepared as described in example 1) using the followingprocedure:

-   -   Trypsinize cells and adjust cell conc. to 1×10E6/ml Hams F12        growth medium    -   Add 500 beads in 500 ul Hams to a 14 ml Nunc tube    -   Add 2500 ul cell suspension 1×10E6/ml Hams w. 5% FCS    -   Leave tube vertically in incubator (37 degrees, 5% CO2) for        16-24 hrs—rock tube gently every 15 min for the first hour    -   Remove medium. Wash loose cells away by gently adding and        removing 4 ml Hams F12 twice (Turn the tube upside down and back        again—as soon as beads have sedimented suck away medium)    -   Add 2 ml Hams F12 w. FCS 10%    -   Incubate o/n at 37 degrees, 10% CO2

Measurement of changes in the cytoplasmic free calcium concentration[Ca²]_(i).

-   -   Beads, now covered with cells, are allowed to sediment for 10        min (no centrifugation needed) and the growth medium is removed        using a pipette.    -   2 ml Krebs Ringer buffer (KRW; KrebsRingerWollheim, pH 7.4: NaCl        0.14 M, KCL 3.6 mM, NaH₂PO₄, H₂O 0.5 mM, MgSO₄, 7H₂O 0.5.mM,        NaHCO₃, 2H₂O 1.5 mM, D-Glucose 6 mM, CaCl₂ 1.5 mM, HEPES 10 mM,        probenecid 2 mM) added 1 uM Fluo-4 (Molecular Probes        F-14201)+0.02% Pluronic (Molecular Probes F-127) per 500 beads        is added, mixed gently and cells/beads are incubated at 37° c.        for 30 min.    -   Beads are hereafter washed w. 5 ml KRW/500 beads ×2 by allowing        sedimentation for 10 min between each wash. The Fluo-4 loaded        cells are now ready for detection of changes in [Ca²⁺]_(i).

The fluorescence is monitored in a fluorescence plate-reader (PolarstarOptima from BMG Labtech, Germany) equipped with a flash Xenon blitz lampand 490±5 nm excitation filter and 510±5 nm emission filter. The platereader is furthermore equipped with a dispenser allowing injection ofagonist.

The measurement can equally well be performed on a microscope equippedwith a fluorescence illuminator (E.g. HBO 100 W lamp) and 480/30 nmexcitation filter, 505 nm LP dicroic mirror and 535/40 nm emissionfilter.

Approx. 50 beads covered with BHK cells expressing the M1 receptor nowloaded with the fluorescent Ca++ indicator Fluo-4 are pipetted into eachwell of a 96 well plate. The plate is placed in the fluorescence platereader and Carbacol 1 uM is injected resulting in an increase influorescence for negative control beads.

This assay can be used to screen for Carbacol inhibitors. Positive hitsare compounds that inhibit the Carbacol (muscarinic M1 agonist)

FIG. 12 shows the intracellular Ca++ mobilization in BHK-M1 cells onbeads treated with Carbamylcholin 100 uM versus control (buffer).

Example 14

Gs Coupled Receptor (MC4R): Agonist Assay (Cre-GFP Reporter AssayDetected with Fluorescence Plate Reader or Fluorescence ImagingEquipment)

Cre-GFP:

Cre-GFP is commercially available from clontech (pCre-d2eGFP) The vectorcontains three copies of Cre-binding sequence fused to a TATA-likepromoter. The vector is holding a neomycin resistance gene. A map of thevector is shown in FIG. 3.

MC4R:

PCR amplified MC4R encoding DNA is introduced into the gateway EntryVector (pENTR) by topoisomarase-mediated ligation. The DNA issubsequently recombined into Destination Vector pDEST12.2.(pDEST12.2MC4R)

Cell Line Establishment:

U2OS cells are transfected with pDEST1.2.2MC4R using standard procedurefor Fugene6 transfection. Cells are put under G418 selection for 4 weeksto obtain a cell line stably expressing the MC4R.

The U2OS cell line stably expressing the human MC4R (melanocortin4receptor) is further transfected with Cre-GFP the day before culturingthem on PEGA beads displaying adhesion peptide and respectively 1)Negative control (PEGA beads with adhesion peptide, but no librarycompound), 2) Positive control (PEGA beads of ex-ample 2) and 3) Librarycompounds. The three cultures are handled separately in each theirculture flask.

Bead/Cell Preparation:

Cells are trypsinized and mixed with the PEGA beads in growth medium(DMEM containing 10% FCS, in the proportion 4000 cells/bead and app. 50ml growth medium/5000 beads.

1) Positive control: 50 ml Growth medium+5000 positive controlbeads+2×10E7 cells.2) Negative control: 50 ml Growth medium+5000 negative controlbeads+2×10E7 cells.3) Screening library (eg. 100,000 compounds): 1000 ml Growthmedium+100.000 library beads+4×10E8 cells

The three culture flasks are placed on a Magnetic stirring platform(Techne) designed for cell culture in suspension and incubated at 37°,5% CO2 for 16-24 hrs using spinning interval 30 rpm, 3 min stirring, 10min pause.

Beads, now covered with cells, are allowed to sediment for 10 min (nocentrifugation needed) and the growth medium is removed using a 50 mlpipette. 10 ml 99% EtOH per 5000 beads is added, mixed gently and leftfor 15 min. Beads are washed w. 10 ml PBS/5000 beads ×3 by allowingsedimentation for 10 min between each wash. Cells are now preserved andfixed to the beads

Plate Reader Assay:

Control beads as well as library beads are seeded in 384 well blackplates (eg. Nunc) with clear bottom app. 20 beads per well. Positive andnegative controls are placed in dedicated wells in 2 times 4 replicatesin each end of the plate. Negative control=20 negative control beads,positive control=one positive control bead+19 negative control beads.The plates are measured in a fluorescence plate reader (PolarStar Optimafrom BMG) using 490+-6 nm excitation filter and 510+-5 nm emissionfilter. Positive control wells are used to determine Smax (maximumresponse)=100% activity and negative control wells to determine 5 min(minimum response)=0% activity. Beads from wells showing activity >30%are collected in a tube for reseeding in a new 384 well plate, this timehaving one bead per well. Smin=one negative control bead and Smax=onepositive control bead. Read plates in plate reader and identify hitsbeads using same procedure as described above.

Image Acquisition Analysis:

Control beads as well as library beads are seeded in 384 well blackplates (eg.

Nunc) with clear bottom app. 20 beads per well. Positive and negativecontrol beads are placed in dedicated wells in 2 times 4 replicates ineach end of the plate. Negative control=20 negative control beads,positive control=one positive control bead+19 negative control beads.Plates are placed on a microscope (Zeiss Axiovert 200M) equipped withfilters allowing fluorescence imaging of eGFP (excitation: 490 nm,emission: 510 nm), 10× objective and motorized stage. One image isacquired for each well followed by image analysis (Metamorph) foridentification of hit beads (green). Beads from hit wells are seeded ina new 384 well plate this time having one bead per well. Smin=onenegative control bead and Smax=one positive control bead. Imageacquisition and analysis described above is repeated and final hit beadsare identified.

Alternatively, approximately 5000 beads are seeded into Lab-TechChambered Coverglass System (#155361; Nalge Nunc INternational), imagingacquisition analysis is performed using the fluorescence equipmentdescribed above, and individual beads that display the requiredfluorescence properties are isolated using a micromanipulator system(Eppendorf Injectman NK). This method is preferred.

Example 14a Gs Coupled Receptor (MC4R) Agonist Screening (Cre-YFPReporter Assay Detected Using a Fluorescence Microscope)

pCre-d₂YFP:

A 732 bp of EYFP fragment from pd2EYFP-1 (Clontech Cat.#6912-1) isligated to a 3.5 kb fragment from pCRE-d2EGFP (Clontech Cat #6034-1).Both fragments are excised from the two vectors by a common restrictionenzyme digestion.

MC4R:

PCR amplified MC4R encoding DNA is introduced into the gateway EntryVector (pENTR) by topoisomarase-mediated ligation. The DNA issubsequently recombined into Destination Vector pDEST12.2.(pDEST12.2MC4R)

Cell Line Establishment:

Hek293 cells are co-transfected with pDEST1.2.2MC4R and Cre-YFP (usingstandard procedure for Fugene6 transfection) and cells are cultured onPEGA beads displaying adhesion peptide and respectively 1) Negativecontrol (PEGA beads with adhesion peptide) and 2) Positive control (PEGAbeads of example 2) by mixing appr. 400 beads with 400,000 cells in 1 mlHams F12 medium containing 10% FCS in a 1.8 ml Eppendorf tube. Tubes areshaked gently every 15 min for 2 hrs. Cells/beads are incubated in a CO2incubator (5% CO2, m 37 degrees) for 20 hrs. The level of CRE-YFPexpression was detected using a Zeiss Axiovert 200M microscope equippedwith appropriate filters for YFP detection (Excitation 500/20 nm,Dicroic 515 LP EM 535/30).

Higher signal was observed for the Hek293 cells compared to the U2OS,why Hek293 were used for further experiments (see FIG. 13).

Library Screening: Synthesis of Library- and Control Beads:

Two libraries were synthesized according to examples 6a and 6b. Controlbeads were synthesized as described in example 5a section “Synthesis ofadhesion peptide”.

Cell Line Establishment:

Hek293 cells were co-transfected with pDEST1.2.2MC4R and pCRE-d2EYFP andput under G418 selection for 3 weeks. Hereafter cells were FACSorted(Fluorescence Activated Bead sorted) for high YFP expression afterstimulation with aMSH 100 nM and 0.4 uM TSA (Tricostatin A) for 20 hrs.Cells were propagated and subcultured for 2 month and FACSorted againfor high aMSH/TSA induced YFP expression.

Cell/Bead Preparation:

Cells were cultured on respectively 1) Negative control beads (preparedas described in example 1), 2) Positive control beads (prepared asdescribed in example 2) and Library beads (prepared as described inexample 6b). Each batch of beads was handled separately.

Cells were trypsinized and mixed with Negative control beads/Positivecontrol beads/Library beads in growth medium (Hams F12 containing 5%FCS):

Control Beads

-   -   Add 500 beads in 500 ul Hams to a 14 ml Nunc tube    -   Add 2500 ul cell suspension 1×10E6/ml Hams w. 5% FCS    -   Leave tube vertically in incubator (37 degrees, 5% CO2) for        16-24 hrs—rock tube gently every 15 min for the first hour    -   Remove medium. Wash loose cells away by gently adding and        removing 4 ml Hams ×2 (Turn the tube upside down and back        again—as soon as beads have sedimented suck away medium)    -   Add 2 ml Hams w. FCS 5% and TSA (Tricostatin A) 0.4 uM    -   Incubate o/n at 37 degrees, 5% CO2    -   Decant beads to a 1 well Lab-Tek Chambered Coverglass (#155361)

Library Beads

-   -   Add 10,000 beads in 5 ml Hams to a 50 ml Nunc tube    -   Add 25 ml cell suspension 2×10E6/ml Hams w. 5% FCS    -   Leave tube vertically in incubator (37 degrees, 5% CO2) for        16-24 hrs—rock tube gently every 15 min for the first hour    -   Remove medium. Wash loose cells away by gently adding and        removing 25 ml Hams ×2 (Turn the tube upside down and back        again—as soon as beads have sedimented suck away medium)    -   Add 25 ml Hams w. FCS 5% and TSA (Tricostatin A) 0.4 uM    -   Incubate o/n at 37 degrees, 5% CO2    -   Decant beads to 2×1 well Lab-Tek Chambered Coverglass (#155361)

Hit Identification and Isolation:

The LabTek one well chambered coverglass was placed on a Zeiss Axiovert200 fluorescence microscope equipped with filters optimal for YFPfluorescence. The microscope was further more equipped with amicromanipulator (Eppendorf Transferman NK2)) capable of picking outsingle beads. Using 10× objective chambers were scanned for positive hitbeads, which appeared as green dotted beads caused by cells expressingCRE-YFP. Positive and negative control beads were used to set cut offfor positive hit beads. Such hit beads were picked out using themicromanipulator for further test in specificity assay (Receptorinternalization) before final structure elucidation.

Microscope detection was preferred for this screening campaign. Thethroughput of this microscope-based method was app. 40,000 beads perday. 90,000 beads were screened in totally. 35 hits were identified andisolated, 15 were structure elucidated and resynthesized.

Signal obtained from a sub-fraction of identified hits is shown in thegraph of FIG. 14.

Structure Elucidation and Resynthesis:

Hit beads are structure elucidated using the method described in example15. As an illustrative example identification of hit designatedTEN-636-33-26 is described om example 15b. Other hits may be identifiedusing a similar method.

Specificity Screening:

Hits are tested for MC4 receptor specificity using a Hek 293 cell linestably expressing the MC4R-GFP as described in example 7a under cellline establishment. Cells are seeded in Hams F12 w. 10% FCS in an 8 wellLab-tek Chambered Cover-glass to give 75% confluency 24 hrs afterseeding. Cells are challenged with hit compounds for 30 min at 37degrees. The chamber is placed on a Zeiss Axiovert 200M equipped withfilters suited for GFP fluorescence and cells are inspected for MC4R-GFPinternalization using image acquisition (20×) followed by imageanalysis. Negative and positive controls are Hams F12 respectively αMSH100 nM.

Selectivity screening β2-adrenergic receptor (β2-AR-GFP Internalization:

Hits are further tested for receptor selectivity using a Hek293 cellline stably expressing the β2-adrenergic receptor fused to GFP(β2-AR-GFP). Cells are tested as described above for “Specificitydetermination”.

MC4R specific hits are those showing a positive response in CRE-YFPreporter assay, a positive response in the MC4R-GFP internalizationassay (specificity) and a negative response in β2-AR-GFP internalizationassay (selectivity).

Example 15 Identification of Compound

Once a resin bead is selected, the library compound comprised within thebead is identified. The selected, single resin bead is treated with 0.1M aqueous NaOH (10 μL) in a 0.5 mL Eppendorf tube overnight, thendiluted with CH₃CN (20 μL), before filtering the solution, therebyproviding a sample for ES MSMS analysis on a Micro-Mass QTOF GlobalUltima mass spectrometer (mobile phase 50% CH₃CN (aq), 0.1 μL/min)employing a linear ramping of the collision energy. The spectra areanalyzed by generating the exact mass differences between fragment ionsand tabulated to provide the fragmentation pathway and from that thestructure of the compound released from the selected bead is elucidated.

Example 15b Identification of Hit TEN-636-33-26 (From Library Preparedas Described in Example 6b) from GPCR Assay

Hits selected in the GPCR assay call for unambiguous structureassignment. High purity of compounds generated on the solid supportduring library synthesis are preferred for single bead analysis. Forexample for the chemistry utilized in Example 6a-b (indsaet evt. ref tilscaffold-patent), it has demonstrated that quantities of materialcleaved from single synthesis and library beads can be analyzed andidentified by QTOF MSMS (ES). The signal arising from the molecular ion(M+H) is first detected, and MSMS is subsequently carried out to obtaina specific fragmentation pattern.

A hit (bead) selected in the assay (FIG. 15 a) is carefully washed with10% TFA (aq) and MiliQ water by successive rounds of decantation. Thebead is placed in a 1.5 mL Eppendorf tube and treated with 0.1 M NaOH (3mL) prepared in the usual way from solid NaOH pellets and MiliQ water.Hydrolysis is effected during 2 h to 24 h in a sealed tube. Afterhydrolysis, 0.1 M HCl (3 mL) is added to neutralize the alkalinecleavage mixture, followed by addition of MiliQ water (40 μL). Prior toloading, the wells of the OASIS elution plate was carefully washed with3×CH₃CN:H₂O (4:1, 0.1% HCOOH), then 3×H₂O (0.1% HCOOH), and 3×H₂O. Theselected well is loaded with the sample solution by applying gentlesuction. Salts are then washed out with water (70 μL) and 0.1% HCOOH (70mL), before eluting the compound with CH₃CN:H₂O (4:1, 0.1% HCOOH) (200μL). The eluent is removed on the speed vac, and the resulting residueis taken up in CH₃CN:H₂O (19:1, 0.05% TFA) (50 μL) before analysis byQTOF LC/MSMS (FIG. 15 b).

Identification of Compound (from Libraries Described in Examples 6a-b)

Libraries containing heterocyclic scaffolds attached to peptidesequences (see Example 6a-b) are very well applicable to single-beadMSMS analysis (material cleaved from single library beads). Thiscompound class generally displays a high propensity to afford uniquedetectable fragments corresponding to the heterocyclic scaffold corestructures. Recognizing this peak in MSMS analysis of the anticipatedmolecular ion, and relying on the general tendency of peptides tofragment at amide bonds, a predictable fragmentation pattern is emerging(See FIG. 16), since each randomized position of amino acids is given bytheir unique masses (with the pairs of leucine/isoleucine andglutamine/lysine as the only exceptions).

Example 16 Multiple GPCR Receptors β2-Adrenergic Receptor (β2AR)-GFP(for Internalization Studies):

Hek293 cells are transfected with β2-adrenergic receptor (β2AR)-GFPusing standard procedure for Fugene6 transfection. Cells are put underzeocin selection for 4 weeks to obtain a cell line stably expressing12-adrenergic receptor (β2AR)-GFP.

Bead/Cell Preparation

Hek293 cells stably expressing β2AR-GFP are seeded in a Nunc 8 wellchambered coverglass in Hams F12 w. 10% FCS and incubated at 37 degrees,5% CO₂ for 20 hrs.

Cells are stimulated with isoproterenol 100 uM (positive control) andmedium (negative control) for 30 min. Cells are imaged on a ZeissAxiovert 200M fluorescence microscope equipped with optimal filters forGFP.

For negative control the β2-adrenergic receptor (β2AR)-GFP is localizedin the membrane whereas for positive control β2-adrenergic receptor(β2AR)-GFP is localized in intracellular spots as an indication ofreceptor activation and consequently internalization.

Compounds modulating the β2AR can be identified in a similar manner bygrowing above-mentioned cells on resin beads comprising librarycompounds, such as the resin beads used in example 14a.

Abbreviations: HGF: Hepatocyte Growth Factor NGF: Nerve Growth FactorPDGF: Platelet Derived Growth Factor FGF: Fibroblast Growth Factor

EGF: epidermal Growth FactorGH: Growth hormone

TRE: TPA Response Element

SRE: serum response elementCRE: cAMP response elementAcN: acetonitril;Boc: tert-butoxycarbonyl;Bu^(t): tert-butyl;DCM: dichlormethane;DMF: dimethylformamide;Fmoc: 9-fluorenylmethoxycarbonyl;HMBA: 4-hydroxymethylbenzoic acid;Q-TOF MS: quadrupole time-of-flight mass spectrometry;MeIm: N-methyl imidazole;MSNT: 1-(mesitylene-2-sulphonyl)-3-nitro-1H-1,2,4-triazole;NEM: 4-ethyl morpholine;PEGA: polyethylene glycol-polydimethyl acrylamide resin;Pfp: pentafluorophenyl;Pmc: 2,2,5,7,8-pentamethylchroman-6-sulfonyl;RP-HPLC: reversed phase high pressure liquid chromatography;SPPS: solid phase peptide synthesis;TBTU: O-(benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumtetrafluoroborate;TFA: trifluoro acetic acid;Thi: thienylFur: furanylBzThi: benzothienyl

1. A method of identifying a compound modifying at least one cellularresponse, wherein each cellular response is linked to different reportersystems generating detectable outputs, said method comprising the stepsof: (a) Providing multiple solid supports capable of supportingadherence and growth of cells, wherein each solid support is covalentlylinked to one member of a library of test compounds and wherein at leasttwo solid supports comprise different library members; and (b) Attachingcells comprising said reporter system(s) onto said solid support;wherein cells are directly attached to the solid support, and/or atleast 10% of the solid supports comprise cell adhesion molecules as wellas said library member, and cells adhere to said cell adhesionmolecules; and (c) Screening said solid supports for solid supportscomprising cells meeting at least one predetermined selection criterion,wherein said selection criterion is linked directly or indirectly tosaid detectable output; and (d) Selecting solid supports comprisingcells meeting said at least one selection criterion; and (e) Identifyingsaid library member, thereby identifying a compound modifying said atleast one cellular response.
 2. The method according to claim 1, whereinsaid adherence is mediated through a cell adhesion compound coupled tosaid solid support, wherein said cell adhesion compound enables saidsolid support to support growth of cells.
 3. The method according toclaim 1, wherein the solid supports are resin beads.
 4. (canceled) 5.The method according to claim 1, wherein the solid supports are spots orregions on a surface or a plated gel or a membrane.
 6. The methodaccording to claim 2, wherein said cell adhesion compound is a peptidewith an overall positive netcharge.
 7. (canceled)
 8. The methodaccording to claim 6, wherein said cell adhesion compound is selectedfrom the group consisting of SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9, SEQ ID 10, SEQ ID 11, SEQID 12, SEQ ID 13, SEQ ID 14, SEQ ID 15, SEQ ID 16, SEQ ID 17, SEQ ID 18,SEQ ID 19, SEQ ID 20, SEQ ID 21, SEQ ID 22, SEQ ID 23, SEQ ID 24, SEQ ID25, SEQ ID 26, SEQ ID 27, SEQ ID 28, SEQ ID 29, SEQ ID 30, SEQ ID 31,SEQ ID 32, SEQ ID 33, SEQ ID 34, SEQ ID 35, SEQ ID 46, SEQ ID 47, SEQ ID48, SEQ ID 49, SEQ ID 50, SEQ ID 51, SEQ ID 52, SEQ ID 53, SEQ ID 54,SEQ ID 55, SEQ ID 56, SEQ ID 57, SEQ ID 58, SEQ ID 59, SEQ ID 60, SEQ ID61, SEQ ID 62, SEQ ID 63, SEQ ID 64, SEQ ID 65, SEQ ID 66, SEQ ID 67,SEQ ID 68, SEQ ID 69 and SEQ ID
 70. 9. The method according to claim 1,wherein said cellular response is modulation of a signal transductionpathway mediated by a cell surface molecule.
 10. The method according toclaim 9, wherein said cell surface molecule is a G-protein coupledreceptor (GPCR).
 11. The method according to claim 10, wherein said GPCRis selected from the group consisting of GPCR of table
 3. 12. (canceled)13. (canceled)
 14. (canceled)
 15. (canceled)
 16. (canceled)
 17. Themethod according to claim 1, wherein the cellular response is modulationof transcriptional activity.
 18. (canceled)
 19. The method according toclaim 1, wherein the cellular response is change in the intracellularlevel of a compound
 20. (canceled)
 21. (canceled)
 22. The methodaccording to claim 1, wherein the cellular response is relocalisation ofa compound.
 23. (canceled)
 24. (canceled)
 25. (canceled)
 26. (canceled)27. (canceled)
 28. (canceled)
 29. The method according to claim 1,wherein the reporter system is a system endogenous to said cells. 30.(canceled)
 31. (canceled)
 32. (canceled)
 33. The method according toclaim 1, wherein the reporter system comprises a nucleic acid comprisinga nucleotide sequence encoding a detectable polypeptide operably linkedto a response element, the activity of which is modulated by thecellular response.
 34. (canceled)
 35. The method according to claim 9,wherein the reporter system comprises a nucleic acid comprising anucleotide sequence encoding a detectable polypeptide operably linked toa response element selected from the group consisting of cAMP responseelement (CRE) and serum response element (SRE).
 36. The method accordingto claim 9, wherein the reporter system comprises a nucleic acidcomprising a nucleotide sequence encoding a detectable polypeptideoperably linked to transcriptional response element (TRE). 37.(canceled)
 38. (canceled)
 39. The method according to claim 1, whereinsaid detectable polypeptide is selected from the group consisting offluorescent proteins and enzymes.
 40. (canceled)
 41. The methodaccording to claim 1, wherein the reporter system comprises abioluminescent moiety.
 42. (canceled)
 43. (canceled)
 44. The methodaccording to claim 1, wherein one predetermined selection criteria is aquantitative level of said bioluminescence above or below a specificthreshold.
 45. The method according to claim 1, wherein thepredetermined selection criteria is specific localisation of afluorescent signal.
 46. The method according to claim 1, wherein saidcells are selected from the group consisting of mammalian cells. 47.(canceled)
 48. (canceled)
 49. The method according to claim 9, whereinthe cells attached to the resin beads comprise a nucleic acid comprisinga first nucleotide sequence encoding said cell surface molecule operablylinked to a second nucleotide sequence not naturally associatedtherewith directing expression of said first sequence.
 50. The methodaccording to claim 1, wherein at least 100 resin beads comprisingdifferent library members are provided.
 51. (canceled)
 52. The methodaccording to claim 1, wherein the library is selected from the groupconsisting of peptides, glycopeptides, lipopeptides, nucleic acids (DNAor RNA), [oligosaccharides,] chemically modified peptides,glycopeptides, nucleic acids (DNA or RNA) [oligosaccharides,] and smallorganic molecules.
 53. The method according to claim 1, wherein thelibrary is a library of small organic molecules.
 54. The methodaccording to claim 1, wherein compounds modifying at least two cellularresponses are identified, wherein step c) involves screening said resinbeads for beads comprising cells meeting at least two predeterminedselection criterion, wherein each selection criterion is related to adifferent detectable output.
 55. The method according to claim 1,wherein the resin bead comprises or consists of polyethylene glycol 56.(canceled)
 57. A cell adhesion compound selected from either: i) thegroup consisting of peptides of: SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ED 4,SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9, SEQ ID 10, SEQ ID 11,SEQ ID 12, SEQ ID 13, SEQ ID 14, SEQ ID 15, SEQ ID 16, SEQ ID 17, SEQ ID18, SEQ ID 19, SEQ ID 20, SEQ ID 21, SEQ ID 22, SEQ ID 23, SEQ ID 26,SEQ ID 27, SEQ ID 28, SEQ ID 29, SEQ ID 30, SEQ ID 31, SEQ ID 32, SEQ ID33, SEQ ID 34, SEQ ID 35, SEQ ID 46, SEQ ID 47, SEQ ID 48, SEQ ID 49,SEQ ID 50, SEQ ID 51, SEQ ID 52, SEQ ID 53, SEQ ID 54, SEQ ID 55, SEQ ID56, SEQ ID 57, SEQ ID 58, SEQ ID 59, SEQ ID 60, SEQ ID 61, SEQ ID 62,SEQ ID 63, SEQ ID 64, SEQ ID 65, SEQ ID 66, SEQ ID 67, SEQ ID 68, SEQ ID69 and SEQ ID 70 or ii) a peptide comprising at least one D-form aminoacid, said peptide being selected from the group consisting of: SEQ ID1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8,SEQ ID 9, SEQ ID 10, SEQ ID 11, SEQ ID 12, SEQ ID 13, SEQ ID 14, SEQ ID15, SEQ ID 16, SEQ ID 17, SEQ ID 18, SEQ ID 19, SEQ ID 20, SEQ ID 21,SEQ ID 22, SEQ ID 23, SEQ ID 24, SEQ ID 25, SEQ ID 26, SEQ ID 27, SEQ ID28, SEQ ID 29, SEQ ID 30, SEQ ID 31, SEQ ID 32, SEQ ID 33, SEQ ID 34,SEQ ID 35, SEQ ID 46, SEQ ID 47, SEQ ID 48, SEQ ID 49, SEQ ID 50, SEQ ID51, SEQ ID 52, SEQ ID 53, SEQ ID 54, SEQ ID 55, SEQ ID 56, SEQ ID 57,SEQ ID 58, SEQ ID 59, SEQ ID 60, SEQ ID 61, SEQ ID 62, SEQ ID 63, SEQ ID64, SEQ ID 65, SEQ ID 66, SEQ ID 67, SEQ ID 68, SEQ ID 69 and SEQ ID 70.58. A resin bead comprising a cell adhesion compound selected from thegroup consisting of SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5,SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9, SEQ ID 10, SEQ ID 11, SEQ ID 12,SEQ ID 13, SEQ ID 14, SEQ ID 15, SEQ ID 16, SEQ ID 17, SEQ ID 18, SEQ ID19, SEQ ID 20, SEQ ID 21, SEQ ID 22, SEQ ID 23, SEQ ID 24, SEQ ID 25,SEQ ID 26, SEQ ID 27, SEQ ID 28, SEQ ID 29, SEQ ID 30, SEQ ID 31, SEQ ID32, SEQ ID 33, SEQ ID 34, SEQ ID 35, SEQ ID 46, SEQ ID 47, SEQ ID 48,SEQ ID 49, SEQ ID 50, SEQ ID 51, SEQ ID 52, SEQ ID 53, SEQ ID 54, SEQ ID55, SEQ ID 56, SEQ ID 57, SEQ ID 58, SEQ ID 59, SEQ ID 60, SEQ ID 61,SEQ ID 62, SEQ ID 63, SEQ ID 64, SEQ ID 65, SEQ ID 66, SEQ ID 67, SEQ ID68, SEQ ID 69 and SEQ ID
 70. 59. The resin bead according to claim 58,wherein said resin bead comprises polyethylene glycol.
 60. (canceled)61. A method of manufacturing a compound modifying at least one cellularresponse, wherein said method comprises the steps of: a) Identifyingsaid compound by the method according to claim 1 b) Preparing saidcompound by chemical synthesis c) Thereby manufacturing said compound62. A method of modulating the activity of a GPCR receptor comprisingthe steps of a) Providing a compound identified by the method accordingto claim 10 b) Incubating said compound together with cells expressingsaid GPCR c) Thereby modulating the activity of said GPCR
 63. Compoundidentified by the method according to claim 1
 64. A method ofsynthesising a cyclic peptide or peptide mimetic library, comprising thesteps i) Providing a plurality of peptides or peptide mimeticscovalently linked to an azide moiety and an acetylene moeity; and ii)cyclizing said peptide or peptide mimetic through a Cu(I) catalysedreaction between said azide- and said acetylene moiety; and iii) therebyobtaining a library of cyclic peptides or peptide mimetics.
 65. Themethod according to claim 64, wherein each peptide or peptide mimeticare immobilised on a solid support.
 66. The method according to claim64, wherein the solid support is resin beads and each resin beadcomprises only one library member in one or more copies.
 67. A libraryprepared by the method according to claim
 64. 68. The method accordingto claim 1, wherein the library of test compounds is a cyclic peptide orpeptide mimetic library prepared by a method comprising the steps i)providing a plurality of peptides or peptide mimetics covalently linkedto an azide moiety and an acetylene moeity; and ii) cyclizing saidpeptide or peptide mimetic through a Cu(I) catalysed reaction betweensaid azide- and said acetylene moiety; and iii) thereby obtaining alibrary of cyclic peptides or peptide mimetics.
 69. A method ofsynthesising a library of heterocyclic ureas, comprising the steps of i)Providing a plurality of urea containing peptide aldehydes; and ii)Subjecting said urea containing peptides to an intramolecularPictet-Spengler reaction; and iii) Thereby obtaining a library ofheterocyclic ureas
 70. The method according to claim 69, wherein saidurea containing peptide aldehydes are immobilised on a solid support.71. A library obtained by the method according to claim
 69. 72. Themethod according to claim 1, wherein the library of test compounds is alibrary of heterocyclic ureas prepared by a method comprising the stepsof i) providing a plurality of urea containing peptide aldehydes; andii) subjecting said urea containing peptides to an intramolecularPictet-Spengler reaction; and iii) thereby obtaining a library ofheterocyclic ureas
 73. The method according to claim 1, wherein thelibrary of test compounds is a library of heterocyclic compoundsobtained by cyclisation of a peptide aldehyde through an intramolecularPictet-Spengler reaction.